High-magnification Electron Microscopy Research Articles

Study of Crack Closure Effect of Hull Plate under Low Cycle Fatigue

The crack closure phenomenon significantly influences low cycle fatigue (LCF) crack growth. The crack closure theory deems that a crack can grow only when the applied load is greater than the fatigue crack opening and closing loads. The revised crack closure theory proposed in this paper provides a new understanding of crack growth: It is no longer the range of stress intensity factor ΔK that controls the crack growth rate, but the effective stress intensity factor ΔKeff. Therefore, it is of great importance to study the crack closure phenomenon of LCF. A combination of experiments and the finite element method (FEM) was used to study the effect of overload on the crack closure effect, and the study was carried out using compact tensile (CT) specimens made of AH32 steel. The FEM was used to obtain the stress changes near the crack tip and the opening displacement changes in the crack trailing area after a single tensile overload, to study the intrinsic mechanism of overload on crack closure, and to obtain the LCF crack opening and closing loads by the nodal displacement method. The effect of overload on crack morphology was observed by using high-magnification electron microscopy in combination with testing.

Coaxial Synthesis of PEI-Based Nanocarriers of Encapsulated RNA-Therapeutics to Specifically Target Muscle Cells.

In this work, we performed a methodological comparative analysis to synthesize polyethyleneimine (PEI) nanoparticles using (i) conventional nanoprecipitation (NP), (ii) electrospraying (ES), and (iii) coaxial electrospraying (CA). The nanoparticles transported antisense oligonucleotides (ASOs), either encapsulated (CA nanocomplexes) or electrostatically bound externally (NP and ES nanocomplexes). After synthesis, the PEI/ASO nanoconjugates were functionalized with a muscle-specific RNA aptamer. Using this combinatorial formulation methodology, we obtained nanocomplexes that were further used as nanocarriers for the delivery of RNA therapeutics (ASO), specifically into muscle cells. In particular, we performed a detailed confocal microscopy-based comparative study to analyze the overall transfection efficiency, the cell-to-cell homogeneity, and the mean fluorescence intensity per cell of micron-sized domains enriched with the nanocomplexes. Furthermore, using high-magnification electron microscopy, we were able to describe, in detail, the ultrastructural basis of the cellular uptake and intracellular trafficking of nanocomplexes by the clathrin-independent endocytic pathway. Our results are a clear demonstration that coaxial electrospraying is a promising methodology for the synthesis of therapeutic nanoparticle-based carriers. Some of the principal features that the nanoparticles synthesized by coaxial electrospraying exhibit are efficient RNA-based drug encapsulation, increased nanoparticle surface availability for aptamer functionalization, a high transfection efficiency, and hyperactivation of the endocytosis and early/late endosome route as the main intracellular uptake mechanism.

Lack of synaptic protein, calsyntenin-2, impairs morphology of synaptic complexes in mice.

Calsyntenin-2 (Clstn2) is the synaptic protein, which belongs to the superfamily of cadherins, playing an important role in learning and memory. We recently reported that Clstn2 knockout mice (Clstn2-KO) have a deficit of GABAergic interneurons, associated with hyperactivity, deficient spatial memory, and social behavior. Therefore, we sought to characterize morphometric features of the ultrastructure of synaptic complexes of hippocampal and cortical neurons in Clstn2-KO mice, using high magnification electron microscopy. Morphometric analysis revealed a reduction of symmetric (inhibitory) synaptic density, length of synaptic contacts, and postsynaptic density in neurons of Clstn2-KO mice. Moreover, cortical neurons of Clstn2-KO mice were characterized by the predominance of the simplified type of synapses with the emergence of negative curvature of the synaptic zone in Clstn2-KO mice. Notably, presynaptic zones of cortical neurons of Clstn2-KO mice were characterized by the increased number of synaptic vesicles in opposite to the decreased number of synaptic vesicles in the presynaptic zones of hippocampal neurons. Overall, we found that lack of calsyntenin-2 leads to the striking architectonic alterations of synaptic complexes in the mouse brain, disrupting synaptic density, shape, and connectivity.

Nanoscale Structure of Zoned Laurites from the Ojén Ultramafic Massif, Southern Spain

We report the first results of a combined focused ion beam and high-resolution transmission electron microscopy (FIB/HRTEM) investigation of zoned laurite (RuS2)-erlichmanite (OS2) in mantle-hosted chromitites. These platinum-group minerals form isolated inclusions (<50 µm across) within larger crystals of unaltered chromite form the Ojén ultramafic massif (southern Spain). High-magnification electron microscopy (HMEM), high angle-annular dark field (HAADF) and precession electron diffraction (PED) data revealed that microscale normal zoning in laurite consisting of Os-poor core and Os-rich rims observed by conventional micro-analytical techniques like field emission scanning electron microscope and electron microprobe analysis (FE-SEM and EPMA) exist at the nanoscale approach in single laurite crystals. At the nanoscale, Os poor cores consist of relatively homogenous pure laurite (RuS2) lacking defects in the crystal lattice, whereas the Os-richer rim consists of homogenous laurite matrix hosting fringes (10–20 nm thickness) of almost pure erlichmanite (OsS2). Core-to-rim microscale zoning in laurite reflects a nonequilibrium during laurite crystal growth, which hampered the intra-crystalline diffusion of Os. The origin of zoning in laurite is related to the formation of the chromitites in the Earth’s upper mantle but fast cooling of the chromite-laurite magmatic system associated to fast exhumation of the rocks would prevent the effective dissolution of Os in the laurite even at high temperatures (~1200 °C), allowing the formation/preservation of nanoscale domains of erlichmanite in laurite. Our observation highlights for the first time the importance of nanoscale studies for a better understanding of the genesis of platinum-group minerals in magmatic ore-forming systems.

Structural characterization and ab-initio resolution of natural occurring zaccariniite (RhNiAs) by means of Precession Electron Diffraction

The crystal structure of naturally occurring zaccariniite from Loma Peguera (Republica Dominicana) has been studied in Transmission Electron Microscopy (TEM) with variable angle Precession Electron Diffraction (PED) techniques: 0.7° Precession Electron Diffraction Tomography (Precession EDT) for unit cell and Laue class sorting, 0.5° Scanning Precession Diffraction (SPED) for crystal orientation mapping and grain alignment, and high symmetry zone axis 1.2° to 2.2° Zone Axis High Angle Precession Electron Diffraction (ZA high angle PED) for Space Group assessment and supercell information gathering. The natural sample has been prepared into an electron thin lamella by means of Focused Ion Beam (FIB). The analysis of the data has yielded tetragonal cell parameters of 3.86, 3.86, 6.77 Å and space group of P4/nmm for the basic structure, and its constituent atom positions for Ni, As and Rh were determined as well by ab-initio structure resolution method in accordance to the elemental composition of the natural zaccariniite obtained with Energy Dispersive X-ray (EDX) and High Magnification Electron Microscopy (HMEM) analysis. A modulation of the crystal basic structure of 3 by 1 in the basal plane has been reported for the first time on natural occurring zaccariniite.

High resolution three-dimensional reconstruction of fibrotic skeletal muscle extracellular matrix.

Fibrosis occurs secondary to many skeletal muscle diseases and injuries, and can alter muscle function. It is unknown how collagen, the most abundant extracellular structural protein, alters its organization during fibrosis. Quantitative and qualitative high-magnification electron microscopy shows that collagen is organized into perimysial cables which increase in number in a model of fibrosis, and cables have unique interactions with collagen-producing cells. Fibrotic muscles are stiffer and have a higher concentration of collagen-producing cells. These results improve our understanding of the organization of fibrotic skeletal muscle extracellular matrix and identify novel structures that might be targeted by antifibrotic therapy. Skeletal muscle extracellular matrix (ECM) structure and organization are not well understood, yet the ECM plays an important role in normal tissue homeostasis and disease processes. Fibrosis is common to many muscle diseases and is typically quantified based on an increase in ECM collagen. Through the use of multiple imaging modalities and quantitative stereology, we describe the structure and composition of wild-type and fibrotic ECM, we show that collagen in the ECM is organized into large bundles of fibrils, or collagen cables, and the number of these cables (but not their size) increases in desmin knockout muscle (a fibrosis model). The increase in cable number is accompanied by increased muscle stiffness and an increase in the number of collagen producing cells. Unique interactions between ECM cells and collagen cables were also observed and reconstructed by serial block face scanning electron microscopy. These results demonstrate that the muscle ECM is more highly organized than previously reported. Therapeutic strategies for skeletal muscle fibrosis should consider the organization of the ECM to target the structures and cells contributing to fibrotic muscle function.

Thrombin and fibrinogen γ′ impact clot structure by marked effects on intrafibrillar structure and protofibril packing

AbstractPrevious studies have shown effects of thrombin and fibrinogen γ′ on clot structure. However, structural information was obtained using electron microscopy, which requires sample dehydration. Our aim was to investigate the role of thrombin and fibrinogen γ′ in modulating fibrin structure under fully hydrated conditions. Fibrin fibers were studied using turbidimetry, atomic force microscopy, electron microscopy, and magnetic tweezers in purified and plasma solutions. Increased thrombin induced a pronounced decrease in average protofibril content per fiber, with a relatively minor decrease in fiber size, leading to the formation of less compact fiber structures. Atomic force microscopy under fully hydrated conditions confirmed that fiber diameter was only marginally decreased. Decreased protofibril content of the fibers produced by high thrombin resulted in weakened clot architecture as analyzed by magnetic tweezers in purified systems and by thromboelastometry in plasma and whole blood. Fibers produced with fibrinogen γ′ showed reduced protofibril packing over a range of thrombin concentrations. High-magnification electron microscopy demonstrated reduced protofibril packing in γ′ fibers and unraveling of fibers into separate protofibrils. Decreased protofibril packing was confirmed in plasma for high thrombin concentrations and fibrinogen-deficient plasma reconstituted with γ′ fibrinogen. These findings demonstrate that, in fully hydrated conditions, thrombin and fibrinogen γ′ have dramatic effects on protofibril content and that protein density within fibers correlates with strength of the fibrin network. We conclude that regulation of protofibril content of fibers is an important mechanism by which thrombin and fibrinogen γ′ modulate fibrin clot structure and strength.

Graphene-SnO2 nanocomposites decorated with quantum tunneling junctions: preparation strategies, microstructures and formation mechanism.

Tin dioxide (SnO2) and graphene are versatile materials that are vitally important for creating new functional and smart materials. A facile, simple and efficient ultrasonic-assisted hydrothermal synthesis approach has been developed to prepare graphene-SnO2 nanocomposites (GSNCs), including three samples with graphene/Sn weight ratios = 1 : 2 (GSNC-2), 1 : 1 (GSNC-1), and graphene oxide/Sn weight ratio = 1 : 1 (GOSNC-1). Low-magnification electron microscopy analysis indicated that graphene was exfoliated and adorned with SnO2 nanoparticles, which were dispersed uniformly on both the sides of the graphene nanosheets. High-magnification electron microscopy analysis confirmed that the graphene-SnO2 nanocomposites presented network tunneling frameworks, which were decorated with the SnO2 quantum tunneling junctions. The size distribution of SnO2 nanoparticles was estimated to range from 3 to 5.5 nm. Comparing GSNC-2, GSNC-1, and GOSNC-1, GOSNC-1 was found to exhibit a significantly better the homogeneous distribution and a considerably smaller size distribution of SnO2 nanoparticles, which indicated that it was better to use graphene oxide as a supporting material and SnCl4·5H2O as a precursor to synthesize hybrid graphene-SnO2 nanocomposites. Experimental results suggest that the graphene-SnO2 nanocomposites with interesting SnO2 quantum tunneling junctions may be a promising material to facilitate the improvement of the future design of micro/nanodevices.

Autophagy Promotes Oligodendrocyte Survival and Function following Dysmyelination in a Long-Lived Myelin Mutant

The Long-Evans shaker (les) rat has a mutation in myelin basic protein that results in severe CNS dysmyelination and subsequent demyelination during development. During this time, les oligodendrocytes accumulate cytoplasmic vesicles, including lysosomes and membrane-bound organelles. However, the mechanism and functional relevance behind these oligodendrocyte abnormalities in les have not been investigated. Using high-magnification electron microscopy, we identified the accumulations in les oligodendrocytes as early and late autophagosomes. Additionally, immunohistochemistry and Western blots showed an increase in autophagy markers in les. However, autophagy did not precede the death of les oligodendrocytes. Instead, upregulating autophagy promoted membrane extensions in les oligodendrocytes in vitro. Furthermore, upregulating autophagy in les rats via intermittent fasting increased the proportion of myelinated axons as well as myelin sheath thickness in les and control rats. Overall, this study provides insight into the abnormalities described in les as well as identifying a novel mechanism that promotes the survival and function of oligodendrocytes.

Topologically Close-Packed μ Phase Precipitation in Creep-Exposed Inconel 617 Alloy

Two creep-exposed Inconel 617 alloy samples [923 K (650 °C) for 45,000 hours and 973 K (700 °C) for 4000 hours] have been studied using analytical electron microscopy and X-ray diffraction techniques. The thermodynamically predicted equilibrium phases in Inconel 617 alloy were compared with the phases observed which are molybdenum-enriched, topologically close-packed μ-phase, along with precipitates of gamma-prime (γ′), M23C6 and Ti(C,N). The μ-phase precipitates were in the size range 60 to 500 nm (with some larger agglomerates); they were situated both within the grains, along twin and grain boundaries, and near intra- and intergranular carbides. The stacking faults in the μ-phase were observed in high magnification electron microscopy. The precipitation of the μ-phase in these samples is significant for the potential use of this alloy in future generation steam power plants as the appearance of the μ-phase is associated with an increased tendency for cracks and voids to initiate. The μ-phase has not been previously identified in the literature relating to creep or thermal exposure of Inconel 617 alloy.

Membranous Glomerulopathy With Spherules: An Uncommon Variant With Obscure Pathogenesis

Occasional case reports of membranous glomerulopathy described unique subepithelial accumulations of an unusual type of immune deposit composed of spherular structures. The identity of such structures as nuclear pores has been suggested, but not established. We identified a cohort of patients (n = 14, including 1 patient with disease recurrence in an allograft) who presented with nephrotic syndrome and had renal biopsy specimens with light and immunofluorescence microscopic findings characteristic of membranous glomerulopathy. These patients were distinguished by ultrastructural studies that showed glomerular capillary wall accumulations of subepithelial immune deposits composed of uniform spherular structures, while lacking the typical granular electron-dense deposits seen in membranous glomerulopathy. The molecular identity of these spherular structures as nuclear pores was tested by using immunofluorescence microscopy and immunohistochemistry with mouse monoclonal antinuclear pore antibodies (Covance, Princeton, NJ) and anti-Nuclear Pore-O-Linked Glycoprotein (Affinity BioReagents Inc, Golden, CO) antibodies. Measurement of spherular structures by using high-magnification electron microscopy showed an average diameter of 84.5 nm, which correlated well with accepted diameters of nuclear pores (80 to 120 nm). Immunofluorescence microscopy and immunoperoxidase staining with both antibodies showed characteristic beaded staining of nuclear membranes of multiple cell types within normal control kidney, but no staining of immune-type deposits within glomerular basement membranes. These cases form a rare, but distinctive, morphological subclass of membranous glomerulopathy. The antigenic specificity of immune deposits in these cases remains elusive.

Cluster structure of anaerobic aggregates of an expanded granular sludge bed reactor.

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH(4)/g of volatile suspended solids [VSS].day or 1.1 g of CH(4) chemical oxygen demand/g of VSS.day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.

MEDIAN RAPHE (PARAMEATAL) CYSTS OF THE PENIS

Purpose Although median raphe cysts of the penis have been widely reported the pathogenesis is obscure. We describe the microstructural detection of 3 cases of median raphe to study the true pathogenesis. Materials and Methods Three patients presented to our clinic of plastic surgery complaining of abnormal shape of the penis or mass recurrence. High magnification light microscopy and electron microscopy were performed. Results Microstructural study revealed that all 3 cases had similar findings to those of the common urethral mucosa. Conclusions Median raphe cysts are derived from the external paraurethral ducts and contain features that to our knowledge have not been previously reported.

Rough surfaced smooth endoplasmic reticulum in rat and mouse cerebellar Purkinje cells visualized by quick-freezing techniques.

The in vivo structure of the smooth endoplasmic reticulum (ER) was visualized in rat and mouse cerebellar Purkinje cells by using quick-freezing techniques followed by freeze-substitution for ultrathin-sectioning or freeze-fracturing and deep-etching for replicas. High magnification electron microscopy of the ultrathin sections revealed a surprising finding that all the smooth ER are apparently rough surfaced, and heavily studded with a large number of small dense projections. In the soma the smooth ER appears to be similar to its rough counterpart, except that the projections are slightly smaller, less electron dense and less protrusive on the ER membranes than the ribosomes. The projections were short rectangles, 20 x 20 x 6 nm3 in size, covering the cytoplasmic surface of the smooth ER in a checker-board manner where closely packed. After freeze-etching and replication, they appeared to be composed of four subparticles, surrounding a central channel. Thus the projections are very similar to the foot structure (ryanodine receptor) of the sarcoplasmic reticulum. Furthermore, they were distributed exclusively in the ER compartment and were highly concentrated especially in the smooth ER. This localization of the projections coindides with the intracellular distribution of the inositol 1,4,5-trisphosphate (IP3) receptor determined by quantitative immunogold electron microscopy. These findings would suggest that the projections are tetramers of IP3 receptor molecules and could be used as a morphological marker for the smooth ER in Purkinje cells, which spreads from the soma to the axon and dendrite, up to the tips including the spines. In Purkinje cells tubular smooth ER runs freely in a serpentine fashion or are intertwined to make large membraneous tangles without forming cisternal stacks. It is highly probable that the ER cisternal stacks do not exist naturally in Purkinje cells but are formed artificially during the various procedures for chemical fixation.

Inherited demyelinating peripheral neuropathies: relating myelin packing abnormalities to P0 molecular defects.

P0-glycoprotein, the major integral membrane protein of peripheral nerve myelin, is thought to mediate myelination and membrane interactions via its extracellular domain (P0-ED). Molecular modeling of P0-ED has suggested which of its amino acid side-chains may be involved in heterophilic and homophilic adhesions. We previously showed that some of these amino acids are the same ones that are substituted or deleted due to mutations in the human gene for P0 (MPZ), which correlate with certain cases of demyelinating motor and sensory peripheral neuropathies. In the current study, high magnification electron microscopy was used to examine the myelin membrane packing in sural nerve biopsies from patients with MPZ mutations. We found that there were distinguishable ultrastructural phenotypes that could be explained by the alterations in P0-ED. These phenotypes, which were not observed in a control nerve, included widening or irregularity of the extracellular apposition alone (delta Ser34; Arg69Cys), widening at both the extracellular and cytoplasmic appositions (Arg69His), the presence of focal bridges in the widened extracellular space (Arg69His), and a diminished (Arg69Cys) or absence (Arg69His) of staining of the double intraperiod line. Our study, which suggests that the altered P0 is incorporated into the myelin sheath, provides a unique basis for further molecular/ultrastructural correlations between P0-ED structure and myelination irregularities.

Inherited demyelinating peripheral neuropathies: Relating myelin packing abnormalities to P0 molecular defects

P0-glycoprotein, the major integral membrane protein of peripheral nerve myelin, is thought to mediate myelination and membrane interactions via its extracellular domain (P0-ED). Molecular modeling of P0-ED has suggested which of its amino acid side-chains may be involved in heterophilic and homophilic adhesions. We previously showed that some of these amino acids are the same ones that are substituted or deleted due to mutations in the human gene for P0 (MPZ), which correlate with certain cases of demyelinating motor and sensory peripheral neuropathies. In the current study, high magnification electron microscopy was used to examine the myelin membrane packing in sural nerve biopsies from patients with MPZ mutations. We found that there were distinguishable ultrastructural phenotypes that could be explained by the alterations in P0-ED. These phenotypes, which were not observed in a control nerve, included widening or irregularity of the extracellular apposition alone (delta Ser34; Arg69Cys), widening at both the extracellular and cytoplasmic appositions (Arg69His), the presence of focal bridges in the widened extracellular space (Arg69His), and a diminished (Arg69Cys) or absence (Arg69His) of staining of the double intraperiod line. Our study, which suggests that the altered P0 is incorporated into the myelin sheath, provides a unique basis for further molecular/ultrastructural correlations between P0-ED structure and myelination irregularities.

Gold-labelled dystrophin molecule in muscle plasmalemma of mdx control mice as seen by electron microscopy of deep etching replica

The Duchenne muscular dystrophy product 'dystrophin' has been shown to be located at the inner surface of normal muscle plasma membrane. This study was undertaken to visualize the shape of dystrophin molecules and their topographical distribution at the inner surface of murine skeletal muscle plasma membrane. The immunogold electron microscopy of plastic-embedded quadriceps femoris muscles of six mdx mice and six control mice showed the presence of gold particles along the muscle plasma membrane undercoat of all muscle samples from the control mice without any antibody reaction in the mdx mice muscles. The gold-labelled muscles of six mdx and six control mice were quickly frozen by liquid helium in a rapid-freeze apparatus. High magnification electron microscopy of the quick-freeze, deep-etch, rotary-shadow replicas of the gold-labelled muscles demonstrated the presence of dystrophin molecules associated with gold particles at the cytoplasmic surface of mdx control mice. The dystrophin molecules displayed a variety of shapes, such as rods with a reduction in diameter from one end to the other end and/or with the enlargement of their end(s). These dystrophin molecules were incorporated in the meshwork of muscle plasma membrane-associated cytoskeletons.

Observations on the muscle plasma membrane-associated cytoskeletons of mdx mice by quick-freeze, deep-etch, rotary-shadow replica method

The Duchenne muscular dystrophy gene product "dystrophin" is a sarcolemma-associated cytoskeleton which is present just inside the sarcolemma. We investigated the ultrastructure and the relationship of this cytoskeleton to the muscle plasma membrane by immunoelectron microscopy and freeze-etch electron microscopy of liquid helium-frozen fresh muscles. The immunoelectron microscopy of the extensor digitorum longus muscles of six mdx mice and six control mice showed the location of anti-dystrophin antibody along the muscle plasma membrane undercoat of all the muscle samples from the control mice without any antibody reaction in the mdx mice muscles. Fresh extensor digitorum longus muscles of seven mdx and six control mice were quick-frozen in liquid helium in the rapid-freeze device. High-magnification electron microscopy of the deep-etch, rotary-shadow replicas of the frozen muscles showed the network formation and attachment of individual rod-shaped cytoskeletons of variable size to the cytoplasmic surface of the muscle plasma membrane in both mdx mice and control mice. The length of cytoskeletons attached to the muscle plasma membranes was measured and mean length +/- SE in mdx mice and control mice were 98 +/- 4 nm and 101 +/- 3 nm, respectively. Although these values were not statistically different (P greater than 0.1), the distribution frequency of 130-150 nm muscle plasma membrane-associated cytoskeletons was 7.9% in mdx mice versus 14.5% in control mice. Since the predicted length of dystrophin is 125-150 nm, the 130- to 150-nm plasma membrane-associated cytoskeletons of mdx control mice may include dystrophin.

Three-dimensional network of cords: the main component of basement membranes.

Basement membranes were divided into two types: 1) thin basement membranes, such as those of the epidermis, trachea, jejunum, seminiferous tubule, and vas deferens of the rat, the ciliary process of the mouse, and the seminiferous tubule of the monkey, and 2) thick basement membranes, such as the lens capsule of the mouse and Reichert's membrane of the rat. High-magnification electron microscopy was used to examine both types after fixation either in glutaraldehyde followed by postosmication or in potassium permanganate. The basic structure of thin and thick basement membranes was found to be a three-dimensional network of irregular, fuzzy strands referred to as "cords"; the diameter of these cords was variable, but averaged 4 nm in all cases examined. The spaces separating the cords differed, however. In the lamina densa of thin basement membranes, the diameter of these spaces averaged about 14 nm in every case, whereas in the lamina lucida it ranged up to more than 40 nm. Intermediate values were recorded in thick basement membranes. Finally, the third, inconstant layer of thin basement membranes, pars fibroreticularis, was composed of discontinuous elements bound to the lamina densa: i.e., anchoring fibrils, microfibrils, or collagen fibrils. In particular, collagen fibrils were often surrounded by processes continuous with the lamina densa and likewise composed of a typical cord network. Finally, two features were encountered in every basement membrane: 1) a few cords were in continuity with a 1.4- to 3.2-nm thick filament or showed such a filament within them; the filaments became numerous after treatment of the seminiferous tubule basement membrane with the proteolytic enzyme, plasmin, since cords decreased in thickness and could be reduced to a filament, and 2) at the cord surface, it was occasionally possible to see 4.5-nm-wide sets of two parallel lines, referred to as "double tracks." On the basis of evidence that the filaments are type IV collagen molecules and the double tracks are polymerized heparan sulfate proteoglycan, it is proposed that cords are composed of an axial filament of type IV collagen to which are associated glycoprotein components (laminin, entactin, fibronectin) and the double tracks of the proteoglycan.

Rationale for the application of thin, continuous metal films in high magnification electron microscopy.

Various metal films of different thicknesses were deposited on to a particle test specimen and their effects on topographic contrast generation and specimen preservation were determined. Tobacco mosaic virus adsorbed on to thin carbon supports or silicon chips was imaged in TEM or high resolution SE-I SEM at a magnification of 350,000X. Tantalum films of 1-2 nm (average mass) thickness produced best contrasts and prevented volume loss of the particles from electron beam damage. Excessively thick films of 5-10 nm thickness blanketed fine structures and caused severe volume losses. Discontinuous 2 nm thick films of gold or platinum decorated the surfaces, caused a loss in topographic contrasts and induced very high volume losses. Thin continuous metal films were necessary to generate high topographic contrast and to prevent volume loss from beam damage by providing sufficient mechanical stability for small topographic features and increased thermal conductivity of the specimen surface.