Gap-junction quantification in biological tissues: freeze-fracture replicas versus thin sections

The relative efficiency of freeze-fracture replicas versus thin sections for the visualization and quantification of gap junctions in biological tissues has been evaluated. Both methods may underestimate gap-junction number—thin sections for reasons of tissue resolution and freeze-fracture replicas due to the mechanics of the fracturing process. Freeze-fracture misses gap junctions in regions of plasma membrane which are highly contoured, such as the overlapping basal cell processes of Drosophila imaginal wing discs and the interdigitating lateral membrane plications of intercalated discs in cardiac tissue. If the missed gap junctions are relatively large, as they are in both of these examples, freeze-fracture significantly underestimates the total gap-junctional area. Thin sections may miss small gap junctions, but in tissues which contain a range of gap-junction sizes the lost junctions constitute a relatively small fraction of the total junctional area. In neoplastic imaginal wing discs, thin sections were as efficient as freeze-fracture replicas in identifying even the smallest gap junctions. Although freeze-fracture may be the better technique for the qualitative and quantitative documentation of small gap junctions in tissues with relatively flat to gently contoured plasma membranes, and thin sections may be the superior method for gap-junction quantification in tissues containing a range of gap-junctional sizes and highly contoured cellular processes, the data suggest that a combination of the two approaches should be utilized whenever possible.     

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.     

Composite replicas: Methodologies for direct evaluation of the relationship between intramembrane and extramembrane structures

Electron microscopic studies of membrane structure have been facilitated by the recent development of the composite replica technique in which the membrane is freeze-fractured, then inverted and the surface deep-etched and replicated. Examination in stereo of this composite preparation of two replicas with interposed half-membrane and associated surface elements reveals the physical relationship between structures on the surface and within the membrane. Composite replicas of the toad urinary bladder surface demonstrated connections of filamentous glycocalyx elements to intramembrane particles (IMPs). Using a bidirectional shadowing technique, many membrane surface particles also are shown to be associated with underlying IMPs, suggesting that these membrane surface particles are projections of the IMPs above the surface of the membrane. There is evidence that elements whose attachment sites relate to the half-membrane fractured away can be displaced from the membrane surface and lost. Labelling studies using colloidal gold-labelled antibodies were carried out to assess loss of surface mesh from fractured membrane. Gold distributions and amounts were similar in labelled surface replicas, label-fracture specimens, and labelled composite replicas, yet the amount of mesh detected in the composite replicas was less than in the surface replicas. This suggests that while some unlabelled or lightly labelled surface elements can be lost from fractured membranes, ligands stabilize elements and reduce their loss apparently by cross-linking them.     

Imaging intracellular elemental distribution and ion fluxes in cultured cells using ion microscopy: a freeze-fracture methodology

A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the ～0·5 μm lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK₂ rat kangaroo and L5 rat myoblast cultures.     

Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections

Simple and efficient protocols for quantifying immunogold labelling of antigens localized in different cellular compartments (organelles or membranes) and statistically evaluating resulting labelling distributions are presented. Two key questions are addressed: (a) is compartmental labelling within an experimental group (e.g. control or treated) consistent with a random distribution? and (b) do labelling patterns vary between groups (e.g. control vs. treated)? Protocols rely on random sampling of cells and compartments. Numbers of gold particles lying on specified organelle compartments provide an observed frequency distribution. By superimposing test‐point lattices on cell profiles, design‐based stereology is used to determine numbers of points lying on those same compartments. Random points hit compartments with probabilities determined by their relative sizes and so provide a convenient internal standard, namely, the expected distribution if labelling is purely random. By applying test‐line lattices, and counting sites at which these intersect membrane traces, analogous procedures provide observed and expected labelling distributions for different classes of membranes. Dividing observed golds by expected golds provides a relative labelling index (RLI) for each compartment and, for random labelling, the predicted RLI = 1. In contrast to labelling densities of organelles (golds µm^?2) or membranes (golds µm^?1), RLI values are estimated without needing to know lattice constants (area per point or length per intersection) or specimen magnification. Gold distributions within a group are compared by chi‐squared analysis to test if the observed distribution differs significantly from random and, if it is non‐random, to identify compartments which are preferentially labelled (RLI > 1). Contingency table analysis allows labelling distributions in different groups of cells to be compared. Protocols are described and illustrated using worked specimen examples and real data.     

Immunogold labelling in environmental scanning electron microscopy: applicative features for complementary cytological interpretation

We have combined environmental scanning electron microscopy (ESEM) and immunogold labelling (IGL) for the analysis of cell morphology and surface protein detection on human fine needle aspiration, which is processed in thin uniform monolayer (a single layer of cells) on a glass slide by Thin Prep technology. Among scanning electron microscopy techniques, we choose the environmental modality (ESEM) because it allows a slight manipulation of biological samples and an operational time comparable with cytological techniques. Moreover, the Thin Prep technology confirmed a reproducible cell monolayer on glass smear, minimizing problems for the determination of appropriate amount of material per slide. The first experimental data in ESEM-IGL on biological samples with fine needle aspiration Thin Prep, in human thyroid nodules, showed that cells retained their morphology and provided a clear IGL. The optimization of conditions (i.e. vacuum pressure, temperature and relative humidity) confirmed the possibility to observe an immunolabelled biological sample and morphological signal, joined with compositional informations, due to peculiar characteristics of gaseous secondary electron detector in ESEM. The ESEM-IGL and fine needle aspiration Thin Prep could be used in combination for the interpretation of cell morphology and cell surface immunolabelling. Our paper suggests this use as a powerful diagnostic tool in a pre-surgical evaluations, opening a new applicative window for electron microscopy.     

Improved structural detail in freeze-fracture replicas: high-angle shadowing of gap junctions cooled below -170 degrees C and protected by liquid nitrogen-cooled shrouds.

In conventional freeze-fracture replicas, precise complementarity of membrane faces is seldom achieved. In a model system frequently used to evaluate replica quality, vertebrate gap junctions are usually visualized as patches of 8-10 nm P-face intramembrane particles separated by 1-2 nm spaces, while E-face images are represented by 4-6 nm conical pits separated by 5-7 nm wide membrane ridges. However, that disparity in sizes of particles versus pits, as well as the disparity in the widths of the spaces separating particles versus pits, suggests that a significant reduction in complementarity of membrane faces has occurred. In this investigation, a JEOL JFD-9000 freeze-etch machine was modified so that fracturing and replication could be performed at temperatures much colder than commonly employed. With the addition of cryopumps to improve overall vacuum and the installation of optically tight LN2-cooled shrouds surrounding the specimen and the knife, water vapor contamination arising from all sources within the vacuum chamber was reduced substantially, allowing replicas to be made at temperatures down to -185 degrees C. With the specimen at these much colder temperatures, water vapor released by the heat of cleaving was also reduced significantly, providing additional improvement in replica quality. In addition, with higher shadowing angles (greater than 60 degrees) and with the specimen at a much lower temperature, the grain size of the platinum film was noticeably reduced, thereby improving resolution at the molecular level. Under these improved conditions, replicas of rat liver gap junctions revealed that many of the P-face IMPs were tubes 6-7 nm in diameter, but that other IMPs had been stretched and distorted by the fracturing process. More important, however, these high resolution replicas revealed that the replicas of the E-face pits represented three-dimensional molecular casts of the transmembrane proteins comprising the connexon hexamer. This means that before they were replicated, the E-face pits faithfully maintained the shape that the IMPs had before fracturing. These more detailed images revealed a new structure in the center of each E-face pit: a 2-3 nm "peg" that may represent the frozen aqueous matrix of the connexon ion channel that remained after elastic extraction of the surrounding six connexin molecules. Thus, high-angle shadowing at very low specimen temperature under virtually non-contaminating conditions has revealed a new level of detail for membrane structure in freeze-fracture replicas.     

Structural and functional correlates of synaptic transmission in the vertebrate neuromuscular junction

Because vertebrate neuromuscular junctions are readily accessible for experimental manipulation, they have provided a superb model in which to examine and test functional correlates of chemical synaptic transmission. In the neuromuscular synapse, acetylcholine receptors have been localized to the crests of the junctional folds and visualized by a variety of ultrastructural techniques. By using ultrarapid freezing techniques with a temporal resolution of less than 1 msec, quantal transmitter release has been correlated with synaptic vesicle exocytosis at discrete sites called “active zones.” Mechanisms for synaptic vesicle membrane retrieval and recycling have been identified by using immunological approaches and correlated with endocytosis via coated pits and coated vesicles. In this review, available ultrastructural, physiological, immunological, and biochemical data have been used to construct an ultrastructural model of neuromuscular synaptic transmission that correlates structure and function at the molecular level.     

Stereology of cubic particles: various estimators for the size distribution

This paper deals with stereological problems for spatial systems of homothetic particles. In contrast to the traditional technique of estimating the size distribution of the particles from the sizes of section profiles we introduce a new method exploiting size and shape of the observed profiles. The shapes of the section profiles contain additional information about the sizes of the particles. This leads to improved stereological estimation of size distribution. As an example the case is studied when the particles form cubes with random edge length. The method is applied to a two-phase WC–Co cermet structure with cubic particles.     

Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases

An immunoelectron microscopy technique was applied to label Chrysanthemum leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal antibodies at different dilutions and secondary antimouse antibody conjugated with colloidal gold particles of different sizes were used. The monoclonal antibodies demonstrated their specificity against the antigen; immunocytological methods permitted the precise localization and identification of phytoplasmas in thin sections from infected tissues.     

Intercellular junctions and the application of microscopical techniques: the cardiac gap junction as a case model

Intercellular junctions are fundamental to the interactions between cells. By means of these junctions, the activities of the individual cells that make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole. In this review, the work of the authors on one specific type of junction—the cardiac gap junction—is presented as a case model to illustrate how the application of a range of microscopical methods, as part of a multidisciplinary approach, can help extend our understanding of cell junctions and their functions. In the heart, gap junctions form the low-resistance pathways for rapid impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. Gap junctions also form pathways for direct intercellular communication, a function of particular importance for morphogenetic signalling during development. The work discussed demonstrates some of the applications of techniques in electron microscopy, immunocytochemistry and confocal scanning laser microscopy to the understanding of the structural basis of the function of gap junctions in the normal adult heart, the developing heart and the diseased heart. Freeze-fracture electron microscopy of heart tissue prepared by rapid freezing techniques, in which excision-related structural damage to the cells is minimized or avoided, makes it possible to deduce the structure of the functioning gap junction in vivo. Gap junctions in hearts that are beating normally in the living animal until the very instant of freezing consist of connexons (transmembrane channels) organized in a quasicrystalline arrangement, not a ‘random’ arrangement as proposed in the original hypothesis on the structural correlates of gap junction function. Alterations in connexon arrangement occur in response to ischaemia and hypoxia, though the relationship of these to gap-junctional permeability is indirect. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera to synthetic peptides matching portions of the sequence of connexin43, the major gap-junctional protein reported in the heart, were raised. The specificity of the antisera was confirmed by dot blotting, Western blotting and by immunogold labelling of isolated gap junctions. One antiserum (that raised to residues 131–142) was found to be particularly effective as a cytochemical probe. An immunofluorescence labelling procedure for use with confocal scanning laser microscopy was developed to enable the three-dimensional precision mapping of gap junctions through thick slices of cardiac tissue. By exploiting the serial optical sectioning ability of the confocal microscope, we have succeeded in (1) elucidating the organization of gap junctions at the intercalated disc, (2) establishing temporal and spatial patterns of gap-junctional protein expression in embryogenesis that correlate with functional differentiation in subsets of cardiac cells, and (3) demonstrating abnormalities of gap-junction distribution and quantity that may contribute to the genesis of arrhythmias in ischaemic heart disease.     

Nano particles’ behavior in non-Newtonian slurry in mechanical process of CMP

Thin fluid film is thought to be formed between the wafer surface and the pad asperity. Hydrodynamic pressure on the surface asperity is periodically generated when particles are passing through it. Fatigue fracture occurs under the effect of periodic pressure, and the fatigue begins from the top to the bottom of the asperity. The removal rate is calculated based on the energy-balance fracture theory. Particle size and its relative velocity are important parameters that affect the polishing effect. Using the multiphase model and the power–law viscosity model of the slurry, particle’s velocity and its distribution in the slurry are numerically calculated. The results indicate that the slurry film thickness needs to be in the same order of the particle size that the particle can generate effective hydrodynamic pressure to remove the asperity materials.     

非对称截面螺旋流道中微粒的惯性聚焦效应

为实现生物微粒/细胞的精确操控,提出了一种非对称截面螺旋流道结构的惯性微流控芯片.基于仿真和实验的方法,对不同尺寸微粒在微流道中的惯性聚焦行为进行了研究.设计了一种"L"形截面的螺旋流道,采用仿真软件COMSOL研究微流道中的二次流场及微粒的运动轨迹.使用UV激光切割与等离子清洗键合的工艺制作芯片样件,采用高速摄像机和...     

Quantitative measurements of magnetic stray field dynamics of Permalloy particles in a photoemission electron microscopy

By example of a Permalloy particle (40 × 40 μm(2) size, 30 nm thickness) we demonstrate a procedure to quantitatively investigate the dynamics of magnetic stray fields during ultrafast magnetization reversal. The measurements have been performed in a time-resolving photoemission electron microscope using the X-ray magnetic circular dichroism. In the particle under investigation, we have observed a flux-closure-dominated magnetic ground structure, minimizing the magnetic stray field outside the sample. A fast magnetic field pulse introduced changes in the micromagnetic structure accompanied with an incomplete flux closure. As a result, stray fields arise along the edges of domains, which cause a change of contrast and an image deformation of the particles geometry (curvature of its edge). The magnetic stray fields are calculated from a deformation of the X-ray magnetic circular dichroism (XMCD) images taken after the magnetic field pulse in a 1 ns interval. These measurements reveal a decrease of magnetic stray fields with time. An estimate of the lower limit of the domain wall velocity yields about 2 × 10(3) m s(-1).     

Cholinergic synapses in the central nervous system: Studies of the immunocytochemical localization of choline acetyltransferase

Cholinergic synapses can be identified in immunocytochemical preparations by the use of monoclonal antibodies and specific antisera to choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker for cholinergic neurons. Electron microscopic studies demonstrate that the fibers and varicosities observed in light microscopic preparations of many brain regions are small-diameter unmyelinated axons and vesicle-containing boutons. The labeled boutons generally contain clear vesicles and one or more mitochondrial profiles. Many of these boutons form synaptic contacts, and the synapses are frequently of the symmetric type, displaying thin postsynaptic densities and relatively short contact zones. However, ChAT-labeled synapses with asymmetric junctions are also observed, and their frequency varies among different brain regions. Unlabeled dendritic shafts are the most common postsynaptic elements in virtually all regions examined although other neuronal elements, including dendritic spines and neuronal somata, also receive some cholinergic innervation. ChAT-labeled boutons form synaptic contacts with several different types of unlabeled neurons within the same brain region. Such findings are consistent with a generally diffuse pattern of cholinergic innervation in many parts of the central nervous system. Despite many similarities in the characteristics of ChAT-labeled synapses, there appears to be some heterogeneity in the cholinergic innervation within as well as among brain regions. Differences are observed in the sizes of ChAT-immunoreactive boutons, the types of synaptic contacts, and the predominant postsynaptic elements. Thus, the cholinergic system presents interesting challenges for future studies of the morphological organization and related function of cholinergic synapses.     

SiC颗粒掺杂对激光直接成形Al2O3陶瓷裂纹敏感性的影响

为抑制激光直接成形Al2O3陶瓷过程中的裂纹,利用SiC未熔颗粒的增韧原理,在Ti-6Al-4V合金基底上进行添加SiC颗粒的Al2O3同轴送粉激光直接成形实验,分析了激光直接成形Al2O3+ SiC复相陶瓷的可行性以及成形件裂纹敏感性的影响因素.利用光学显微镜观察薄壁成形试样的裂纹扩展、显微组织和两相结合情况,并使用X射线衍射仪(XRD)进行相分析.结果表明:SiC颗粒可在激光直接成形Al2O3+SiC陶瓷中起到抑制裂纹的作用,并可形成各成分结合良好,无明显化学反应,含有较完整SiC未熔颗粒的复相陶瓷材料.单因素实验显示:SiC比例f、激光功率P、扫描速度v和送粉率n对裂纹敏感性均有显著影响,最后采用工艺参数:f =10％(重量百分比)、P=186 W、v=300 mm/min及n=1.78 g/min成形了裂纹敏感性低,无明显缺陷的长×高×厚约为17 mm×6 mm×2 mm的薄壁件.     

A comparison of some methods for measuring the size distribution of two-dimensionally clustered latex particles in TEM images using mathematical morphology

The particle size distribution of polymer latexes of, for example, polybutadiene can be measured on transmission electron micrographs using image analysis methods. By taking suitable measures in preparing the sample it can be ensured that the image only consists of projections of two-dimensional clusters of adhering latex spheres. For the deagglomeration of these clusters a number of methods based on mathematical morphology are discussed. Speed and accuracy of the methods were tested on a personal-computer-based image analysis system and compared to manual measurements. Repeated openings or erosions applied to a binary image are relatively fast global methods with a low accuracy due to the fact that particle diameters are measured in whole numbers of pixels; only the horizontal diameters of rectangular pixels are measured. With an iterative method the individual components of the clusters are found by repeated conditional thickening of the erosion kernels. The method is accurate but very slow due to the large number of processing steps. The watershed method as applied to the original grey-scale transmission electron micrograph makes use of the sharp local maxima which arise at the interface of the projection of two adhering particles. This method is as accurate as the iterative method, but about eight times as fast.     

Dry fracturing and cutting for techniques for scanning electron microscopy of poly-vinyl chloride particles: Application to internal structure observations

Two methods for examining the internal structure of poly-vinyl chloride (PVC) particles are described. Very small PVC particles, polymerized by the emulsion process, were mounted on an aluminium adhesive tape and pressed with a similar tape. Both tapes were then pulled apart so that the specimen was broken in two fractions, which were observed by scanning electron microscopy. On the other hand, bigger PVC particles manufactured by following the suspension polymerization process, were frozen and hand sectioned with a razor blade under liquid nitrogen vapor. The results were highly satisfactory. These methods were easy to implement, the cost of materials for sample preparation was negligible, and they offered the ability to obtain multiple information from a single sample.     

Friction and wear of polyphenylene sulfide composites filled with micro and nano CuO particles in water-lubricated sliding

The tribological behavior of polyphenylene sulfide (PPS) composites filled with micro and nano CuO particles in water-lubricated sliding condition were studied. Pin-on-disk sliding tests were performed against a steel counterface of surface roughness 0.09–0.11 μm. The lubrication regimes were established from friction data corresponding to various combinations of loads and sliding speeds. Later experiments were performed using the sliding speed of 0.5 m/s and contact pressure of 1.95 MPa, which corresponded to boundary lubrication regime. Micro CuO particles as the filler were effective in reducing the wear of PPS but nano CuO particles did not reduce wear. The steady state wear rate of PPS-30 vol.% micro CuO composite was about 10% of that of unfilled PPS and the coefficient of friction in this case was the lowest. The examination of the topography of worn pin surfaces of nano CuO-filled PPS by SEM revealed grooving features indicating three-body abrasion. The transfer films formed on the counterfaces during sliding were studied by optical microscopy and AFM. The wear behavior of the composites in water-lubricated sliding is explained using the characteristics of worn pin surfaces and transfer films on the counterface.     

Endopolygalacturonase-gold complex: Parameters influencing the labelling of pectic acid in the cell walls of flax hypocotyl

An enzyme/colloidal-gold complex was prepared using a purified endopolygalacturonase (endoPGH, E.C. 3.2.1.15). Gold particles with a radius of 4·8 nm were prepared. To stabilize the gold particles, it was necessary to add a minimal amount of enzyme (approximately 0·5 μg) at a pH higher than its isoelectric pH. Under these conditions, the number of protein molecules adsorbed per gold particle was approximately one. The parameters influencing the diffusion of the endoPGH-gold complex to its target molecules were screened and tested through quantitative approaches: (i) the labelling efficiency q was calculated according to the equation for the diffusion of gold markers from the bulk solutions to target molecules and (ii) the labelling density N/S was measured by counting gold particles on electron micrographs. The highest value of N/S was obtained by determining the optimal values for the parameters outlined in the equation for q. To obtain the highest value of N/S, it was not necessary to choose values for these parameters which gave high values for q. The specificity of the probe towards homogalacturonic acid blocks was established through controls. The localization of these blocks was illustrated throughout the walls of different kinds of flax seeding cells.