Freeze-fractured Specimens Research Articles

The Effect of Poly(Butylene Adipate-co-Terephthalate) on Crystallization Behavior and Morphology of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)

The effects of poly(butylene adipate-co-terephthalate) (PBAT) on crystallization behavior and morphology of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were studied to provide the useful information to control and improve PHBV processing. PHBV were blended with 15, 30 and 50 wt% PBAT by twin screw extrusion and these were subsequently compared to unblended PHBV. The rate of crystal development determined from differential scanning calorimetry (DSC) at 120 °C showed that the incorporation of PBAT retarded the crystal growth rate. Moreover, the crystal structure of polymer blends was examined by X-ray diffraction (XRD) and the results revealed that PBAT did not affect the crystal structure of PHBV. The responses of the melt-crystallized PHBV to different quantities of PBAT were recorded by polarized optical microscopy (POM). The results demonstrated that the size of spherulite dramatically increased when 15 wt% PBAT was added and the shape of spherulite was imperfect when PBAT reached 30 wt%. The morphologies of PHBV and its blends on the freeze-fractured specimens were exposed using scanning electron microscopy (SEM). The SEM images revealed the phase separation of PHBV/ PBAT blends in any composition. The morphology of 15 and 30 wt% PBAT presented droplet in matrix morphology and changed to co-continuous morphology at 50 wt% PBAT.

Effect of Calcium Carbonate on Crystallization Behavior and Morphology of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)

The effects of calcium carbonate (CaCO3) concentration on crystallization behaviors and morphology of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were investigated. Composites of PHBV with CaCO3 were prepared with filler loadings of low (5%wt) and high concentration (20%wt) and these were subsequently compared to unloaded PHBV. The morphologies of PHBV composites on the freeze-fractured specimens were examined using scanning electron microscopy (SEM). The SEM images revealed that increasing concentration of CaCO3 resulted in agglomeration. This agglomeration might affect crystal growth rate and mechanism. The crystal growth behavior of melt-crystallized PHBV with different amounts of CaCO3 was studied by polarized optical microscopy (POM), while the crystal structure was examined by X-ray diffraction (XRD). The rate of crystal growth determined from POM at selected crystallization temperatures revealed that the addition of a small amount of CaCO3 accelerated crystal growth rate, whereas excess amount of CaCO3 had the opposite effect. The POM images were also used to illustrate the change of crystal growth process presence of CaCO3. The unloaded PHBV clearly showed nucleation and growth mechanism, while PHBV composites displayed nucleation and then combination of crystals during the growth process. However, CaCO3 did not affect the crystal structureof any PHBV composite as observed by XRD. Molecular weight determination via gel permeation chromatography (GPC) indicated that there was no significant difference among PHBV composites.

An MBoC Favorite: Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling

In celebration of MBoC’s first 20 years, members of the Editorial Board, members of the ASCB Council, and others comment on their favorite MBoC papers from the past two decades. This article is the MBoC paper most cited by my group, and so it is clearly our MBoC favorite. The authors revived an electron microscopy technique in which membrane molecules fixed on the platinum replicas of quickly frozen and freeze-fractured specimens were immunogold labeled and applied this technique to observe the two-dimensional distribution of gangliosides GM1 and GM3, putative raft molecules in the plasma membrane. The authors were the first to show that GM1 and GM3 form cholesterol-dependent clusters (GM1 cluster size <100 nm) and that these clusters do not coincide. These results indicate the presence of various raft domains with different compositions and the mechanisms for assembling the same species of gangliosides. They were, and still are, exciting data for scientists interested in raft domains, because they clearly indicate that molecule-specific, homophilic interactions, in addition to more general, cholesterol-dependent raft-lipid interactions, are required for generating certain raft domains in the plasma membrane. This work changed the concept of raft domains.

Gangliosides GM1 and GM3 in the Living Cell Membrane Form Clusters Susceptible to Cholesterol Depletion and Chilling

Presence of microdomains has been postulated in the cell membrane, but two-dimensional distribution of lipid molecules has been difficult to determine in the submicrometer scale. In the present paper, we examined the distribution of gangliosides GM1 and GM3, putative raft molecules in the cell membrane, by immunoelectron microscopy using quick-frozen and freeze-fractured specimens. This method physically immobilized molecules in situ and thus minimized the possibility of artifactual perturbation. By point pattern analysis of immunogold labeling, GM1 was shown to make clusters of <100 nm in diameter in normal mouse fibroblasts. GM1-null fibroblasts were not labeled, but developed a similar clustered pattern when GM1 was administered. On cholesterol depletion or chilling, the clustering of both endogenous and exogenously-loaded GM1 decreased significantly, but the distribution showed marked regional heterogeneity in the cells. GM3 also showed cholesterol-dependent clustering, and although clusters of GM1 and GM3 were found to occasionally coincide, these aggregates were separated in most cases, suggesting the presence of heterogeneous microdomains. The present method enabled to capture the molecular distribution of lipids in the cell membrane, and demonstrated that GM1 and GM3 form clusters that are susceptible to cholesterol depletion and chilling.

3P294 クライオ原子間力顕微鏡による凍結割断試料の液浸観察(バイオイメージング,ポスター発表,第45回日本生物物理学会年会)

3P294 クライオ原子間力顕微鏡による凍結割断試料の液浸観察(バイオイメージング,ポスター発表,第45回日本生物物理学会年会)

Explorative statistical analysis of planar point processes in microscopy

Basic methods of explorative statistical analysis for stationary and isotropic planar point processes are briefly and informally reviewed. At the explorative level, planar point patterns may be characterized in terms of the intensity, the K-function and the pair correlation function. These second-order functions enable one to classify a given point process as completely random, clustering or repulsive. The repulsive behaviour may be quantified by an estimate of the hard-core distance. In the exploratory approach, the statistics are essentially free from model assumptions. Second-order spatial functions have been estimated to characterize genuine planar point processes in the macroscopic domain, for example in forestry, geography and epidemiology. For light microscopy and transmission electron microscopy, two situations are distinguished, which may be summarized as the genuine planar case and the stereological case. In the genuine planar case, a direct interpretation of the results of spatial statistics is feasible. Here, monolayers in cell culture, intramembranous particles on freeze fracture specimens and amacrine cells of the retina are mentioned as examples. In the stereological case, point patterns are generated by sections through 3D structures. Here the observed point patterns may arise as the centres of sectional profiles of particles, or as centres of sectional profiles of spatial fibre processes. In both situations, exploratory spatial point process statistics allow a quantitative characterization of sectional images for the purposes of group comparisons and classification. Moreover, for spatial fibre processes it has recently been shown that the observed pair correlation function of the centres of the fibre profiles is an estimate of the reduced pair correlation function of the fibre process in 3D. Hence for fibre processes a stereological interpretation of point process statistics obtained from sections is an additional option.

Ultrastructural analysis of differentiation in Legionella pneumophila.

Legionella pneumophila is an adaptive pathogen that replicates in the intracellular environment of fundamentally divergent hosts (freshwater protozoa and mammalian cells) and is capable of surviving long periods of starvation in water when between hosts. Physiological adaptation to these quite diverse environments seems to be accompanied by morphological changes (Garduño et al., p. 82-85, in Marre et al., ed., Legionella, 2001) and conceivably involves developmental differentiation. In following the fine-structural pathway of L. pneumophila through both in vitro and in vivo growth cycles, we have now discovered that this bacterium displays an unprecedented number of morphological forms, as revealed in ultrathin sections and freeze-fracture replicas for transmission electron microscopy. Many of the forms were identified by the obvious ultrastructural properties of their cell envelope, which included changes in the relative opaqueness of membrane leaflets, vesiculation, and/or profuse invagination of the inner membrane. These changes were best documented with image analysis software to obtain intensity tracings of the envelope in cross sections. Also prominent were changes in the distribution of intramembranous particles (clearly revealed in replicas of freeze-fractured specimens) and the formation of cytoplasmic inclusions. Our results confirm that L. pneumophila is a highly pleomorphic bacterium and clarify some early observations suggesting sporogenic differentiation in L. pneumophila. Since morphological changes occurred in a conserved sequence within the growth cycle, our results also provide strong evidence for the existence of a developmental cycle in L. pneumophila that is likely accompanied by profound physiological alterations and stage-specific patterns of gene expression.

Detrusor smooth muscle cells of the guinea-pig are functionally coupled via gap junctions in situ and in cell culture.

Intercellular communication between smooth muscle cells is crucial for contractile behaviour in normal and pathologically altered urinary bladder. Since the study of coupling is difficult in situ, we established cell cultures of bladder smooth muscle cells to analyse coupling mechanisms. Microinjection of Lucifer yellow demonstrated syncytia composed of only a few to several dozen cells. Electron-microscopic examination of freeze-fracture specimens and ultrathin sections revealed that the dye-coupling was based on typical gap junction formation between the cultured smooth muscle cells. Furthermore, we were able to demonstrate gap junctions within the tissue fragments from which the primary cultures were grown. By Western blotting, we found connexin-43-positive protein bands both in native tissue probes from the guinea-pig urinary bladder and in smooth muscle cell cultures. Extracellular electrical stimulation of single cells evoked calcium transients, as visualized by fura-2 ratiofluorimetry. Calcium waves propagated throughout the syncytia with a declining amplitude, showing that the calcium signal was not regenerative. Therefore, the calcium signal was probably transmitted by a diffusible factor. These findings correlated well with the dye-coupling that we found between detrusor smooth muscle cells in situ. The use of smooth muscle cell cultures therefore seems to be a feasible approach for studying coupling behaviour in vitro.

Preservation of Biomembranes by High Pressure Freezing ?

Abstract Sample preparation techniques for electron-microscopy (EM) dictate our perception of the microworld: any structural information which is lost or distorted during preparation can not be regenerated later and might lead to wrong interpretation of the observed micrograph. Cryofixation based procedures for specimen preparation can avoid most of the structural alterations associated with standard techniques based on chemical fixation. The ultrastructure can be represented in a near “native state” thanks to the high time resolution for dynamic cellular events (1). High pressure freezing (2) permits to cryoimmobilize biological samples up to approx. 200μm thick, in contrast to rapid freezing procedures at ambient pressure that are useful to cryoimmobilize samples up to 10-20 μm thick. The actual samplethickness that can be adequately frozen (=without visible damage due to ice crystal formation in freeze-substituted or freeze-fractured specimens) depends on the concentration of naturally occuring substances that exhibit cryoprotective activities.

Effect of lipids on the structure and rheology of gels formed by canine submaxillary mucin

Rheological experiments have shown that canine submaxillary mucin (CSM) forms gels in aqueous solution at low ionic strength and in 6M GdnHCl. Examination of specimens of intact CSM and also its subunits prepared by reduction and carboxymethylation showed that the presence of lipid increases the gel-forming capability, probably as a result of enhancement of the intermolecular hydrophobic interactions. The rheological evidence for gelation is that substantially larger values of the oscillatory storage modulus, G'(ω), and the dynamic complex viscosity, η *(ω), are observed for lipid-containing CSM. TMs is backed up by electron micrographs of freeze fractured specimens, where we observe a network morphology in which the cross-links are formed as a result of non-bonded interactions between a number of CSM chains. The intermolecular interactions responsible for gelation probably involve hydrophobic association between the interdigitated oligosaccharides, and/or between the non-glycosylated regions of the protein core, and can occur even in a highly chaotropic medium (6M GdnHCl). In contrast to previous experiments with porcine submaxillary mucin and human tracheobronchial mucin, which form microphase-separated gels in aqueous solution, CSM solutions undergo macroscopic phase separation into polymerrich (gel) and polymer-poor (sol) phases. These data point to stronger hydrophobic interactions in lipid-containing CSM.

Osteoconduction on, and Bonding to, Calcium Phosphate Ceramic Implants

AbstractCalcium phosphate ceramic blocks of three different densities, porous, intermediate and dense, were prepared by tape casting (total = 18), and implanted in the femora of 9 Wistar rats for 1 to 3 weeks. Following fixation, the tissue was prepared for examination by scanning electron microscopy (SEM) of freeze fractured specimens, or back-scattered electron imaging (BSEI) of polymethylmethacrylate (PMMA) embedded, undecalcified, sections. The results demonstrated that while all ceramics were osteoconductive, the 93 % dense implants showed no evidence of bone bonding as judged by the plane at which specimens separated or cracked during preparation for microscopy. The porous ceramics, on whose surfaces both multinucleated cells and osteoid tissue were observed at all implantation times, exhibited less bone contact than the other two groups. Both SEM and BSEI showed that there was bone growth into the microporosity of both intermediate and porous ceramics, pore size range 1−2μ m, to a depth of about 6μm. The latter corresponds to the thickness of natural bone lamellae. In all implants examined,de novobone formed on the ceramic surface by the initial production of a biological cement-line like interfacial extracellular matrix. The results clearly show that osteoconduction and bone-bonding are distinct mechanistic phenomena. While we assume the former to be a consequence of protein adsorption events culminating in anchorage of osteoblasts to the implant surface, the latter is the result of mechanical interdigitation of the extracellular matrix, produced by osteoblasts, with the microtopography of the implant surface.

Freeze-fracture observations in the L? phase of a swollen surfactant in the vicinity of the L3 and the L1 phase transitions

Freeze-fractured specimens of the Lα phase of a swollen quasi-ternary surfactant (cetylpyridinium chloride/hexanol/brine), have been observed by electron microscopy. The textures show the same features as those oberved by optical microscopy, viz. defects with negative Gaussian curvature (focal conic domains of the usual type) close to the phase boundary with the L3 sponge phase, and defects with positive Gaussian curvature (focal conic domains of second species, mostly spherolites) close to the phase boundary with the L1 micellar phase. We have studied in more detail this latter region : the density of spherolites increases as one approaches the L1-Lα transition, up to the point where they seemingly fill the whole space ; their size distribution obeys an exponential law we explain by a simple thermodynamic model which predicts a transition when the elastic coefficient $\Lambda = 2 K + \bar{K}$ vanishes. Our experimental data yield indeed, with a reasonable precision, a very small value of Λd/kB T for a composition of the surfactant close to the phase boundary.

Membrane structure of parallel-fibre synaptic terminals in the cerebellum of the jaundiced Gunn rat: freeze-fracture and E-PTA study

Parallel-fibre synaptic membranes were examined by freeze-fracture and ethanolic-phosphotungstic acid methods in the cerebellum of homozygous (j/j) Gunn rats with hereditary jaundice. Parallel-fibre synapses with dendritic spines of Purkinje cell were severely affected since many Purkinje cells degenerated during the early postnatal period. Some parallel-fibre synaptic terminals lacked their postsynaptic partners and faced astrocytic processes from 18 days of age to the adult stage. These parallel-fibre terminals contained clusters of synaptic vesicles adjacent to synaptic membranes, and synaptic membranes and synaptic cleft materials were identical to those of parallel fibres with postsynaptic partners, as visualized by conventional electron microscopy with osmium tetroxide postfixation and staining of sections with uranyl acetate and lead citrate. In freeze-fractured specimens, the presynaptic membrane of parallel fibres had diffusely distributed large particles and tiny pits on the P-face and protuberances on the E-face, together representing synaptic vesicle attachment sites. Such vesicle attachment sites were present on the presynaptic membranes of parallel fibres without postsynaptic partners from day 18 to the adult stage. After ethanolic-phosphotungstic acid staining, parallel-fibre terminals displayed presynaptic dense projections, intercleft materials and postsynaptic thickening, but some parallel fibres lacked postsynaptic thickening. These observations suggest that the presynaptic membrane structure of the parallel fibre is preserved, even in the absence of a postsynaptic partner, in j/j cerebella. A mechanism for persistence of presynaptic membrane structures without postsynaptic partners in j/j cerebella is discussed.

Nuclear matrix involvement in sperm head structural organization

In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.

Viewing microemulsions with freeze-fracture transmission electron microscopy

Freeze-fracture and replication is a sample preparation technique for TEM that has been used to investigate microemulsion structure. In previous studies of the water/n-octane/pentaethylene glycol dodecyl ether system, images from identical sample of bicontinuous microemulsions have shown two different morphologies. Here, the authors present a method of preparing freeze-fracture specimens that allows them to see both type of morphologies and understand their origins. In the course of resolving the differences, they have developed a method for contrasting microemulsion structure exposed by a cohesive fracture with that exposed by adhesive fracture at a metal-microemulsion interface.

Cooling rate and ice‐crystal measurement in biological specimens plunged into liquid ethane, propane, and Freon 22

Specimens sandwiched between copper planchettes were plunged up to a depth of 430 mm into coolants used for cryofixation. Hydrated gelatin containing a miniature thermocouple was used to mimic the behaviour of tissue during freezing. Gelatin and red blood cells were used for ice-crystal analysis. Ethane produced the fastest cooling rates and the smallest ice-crystal profiles, and Freon 22 produced the slowest cooling rates and the largest crystal profiles. Smaller crystal profiles were often seen in the centre of the specimens than in subsurface zones. The results show that ethane, rather than propane, should be used for freezing metal-sandwiched freeze-fracture specimens by the plunging method, and probably also in the jet-cooling method. They further suggest that good cryofixation could occur at the centre of thin specimens rather than only at their surfaces. Comparison between theoretical and experimental ice-crystal sizes was satisfactory, indicating that where the experimental parameters can be defined then realistic predictions can be made regarding cryofixation results.

Glycolipid storage material in Fabry's disease: A study by electron microscopy, freeze-fracture, and digital image analysis

The glycolipid storage material in Fabry's disease was studied by electron microscopy of thin-sectioned (TS) and freeze-fractured (FF) specimens. In the kidney all deposits were found to be located in lysosomes, arranged as lamellar stacks. Deposits in the heart consisted of intracytoplasmic concentric whirls or folded lamellar structures. High resolution TS micrographs disclosed various defects in the lamellar structure. For stabilization, such defects require additional amphiphilic, surface-active molecules. These molecules could interact with other cellular constituents. The lamellar periodicity of the deposits in FF specimens was determined by reconstruction of the three-dimensional fracture face by digital image analysis. Homogeneous multilamellar deposits exhibited a periodicity of 14–15 run, contrasting with the conventional estimates of 4–5 nm on TS micrographs. This difference is explained by better preservation of the physiologic hydrated state in FF specimens, with 1 vol of lipids binding 2 vol of water. Inhomogeneous structures with an even higher state of hydration included water lenses between the sheets. The strong hydration obviously contributes to the enlargement of the intracellular glycolipid deposits.

セメント質の線維性基質と層板構造

The purpose of this study was to clarify the relationship between the lamellar structure and the fibrous components of cementum. Freeze-fracture specimens and ground sections of the human molar cementum were treated with acid- and/or alkaline-solutions, then examined by scanning electron microscopy. Both of the layers of Sharpey (extrinsic) fiber and matrix (intrinsic) fiber were distinguished in freeze-fracture specimens and ground sections, but no lamellar structures appeared. For the first time, the lamellae isolated with narrow grooves were clearly observed on the ground sections that were treated with 5% sodium hypochlorite for 60 min followed by 0.5-1.0 M hydrochloric acid for 30-60 sec. In addition to the two fiber-layer types, the mixed fiber layer containing both components of matrix and Sharpey's fibers was classified, and the lamellar arrangement of matrix fibers was also revealed. When the hypochlorite treatment time was prolonged for 120 min, the lamellar structures were evident. Our results show that those treatments may resolve the regions of physiological hypomineralization related to the incremental lines running parallel to the cemento dentinal junction and the lines of discontinuity of the fibrous components.

Lipid polymorphism of model and cellular membranes as revealed by electron microscopy

This review surveys the current state of knowledge relating to lipid polymorphism within both model lipid membrane and cellular membrane systems. Emphasis is placed upon the contribution of data obtained by transmission electron microscopy of freeze-fractured specimens. Some consideration is also given to the other important methods for the study of lipid polymorphism, namely X-ray diffraction and NMR spectroscopy. A detailed account of the different phases found in lipid mixtures within model membranes (bilayer, cubic or tetragonal, tubular or hexagonal) provides the background to the understanding of the factors involved in polymorphic phase transitions. The sequential steps involved in lipid polymorphism are defined from electron microscopical data and are related to the structural changes that can be detected within biological membranes. It is proposed that the fine structural changes detected at the initial stages of polymorphic transition in vivo may be highly relevant in relation to membrane fusion events, to the formation of tight junctions, and even to physiological transport processes. Since the later stages of polymorphic transition generally destroy the permeability barrier of model and cellular membranes, extensive rather than localized phase transition of the lipid bilayer is not at the moment considered to be compatible with cellular viability.

Adhesion at the Interface in Cured Graphite Fiber Epoxy-Amine Resin Composites

The effect of high temperature curing on the interface between unsized or epoxy-sized graphite fiber tow and epoxy-amine resin was examined by scanning electron microscopy of compression and freeze fractured specimens. Little or no adhesion was found between the unsized graphite fiber tows and the epoxy-amine resin on curing at 165°C for 17 hrs. Epoxy-sized graphite fibers showed a similar lack of adhesion between the fiber tows and the epoxy-amine resin at 3 and 17 hrs. cures, although good penetration of the resin into the sized fiber tows had occurred. Interfacial bond strengths for the composites could not be effectively measured by compression fracture of specimens.