Shadowed Replicas Research Articles

Supramolecular Aggregation Processes on Carbon Surfaces Occurring in Bovine Serum Albumin Solutions

The processes of supramolecular aggregation occurring on carbon surfaces in aqueous solutions of bovine serum albumin (BSA) during drying were studied using modern scanning electron microscopy (SEM). The carbon materials studied were highly oriented pyrolytic graphite (HOPG) and glassy carbon (GC). Based on the analysis of SEM images and EDX-scanning element distribution maps, a possible mechanism for the formation of the observed intricate structures on the surface was proposed. The formation of fuzzy lacy structures resembling shadow replicas was explained by relatively strong hydrophobic–hydrophobic interactions of albumin molecules with carbon surfaces.

Transmission Electron Microscope Observation by Surface Replica Method for Layer Undulation in Solid X1 Phase of Banana Molecule

The smectic X1 phase formed from the banana 4-bromo-1,3-phenylenebis[4-(4-n-tetradecanoxyphenyliminomethyl)benzoate] (4Br-P-14-O-PIMB) molecule, possesses a distinct two-dimensional structure with a long-range density modulation of ∼17.5 nm along the layer. The X1 phase is formed upon cooling from the well-known B2 phase, and then its characteristic modulated structure is retained at room temperature. A transmission electron microscopic (TEM) observation was performed for shadowed replica film which imprints the surface structure of the homeotropically aligned X1 phase. The modulation was detected as clear stripes with periodicities ranging from 15 to 18 nm, which agree with the modulation length determined by X-ray measurement. The results confirm that the periodicity along the layer is not due to the simple density modulation occurring within a smooth layer, but can be explained by the layer undulation that creates the crests and troughs.

Molecular organization of Mason-Pfizer monkey virus capsids assembled from Gag polyprotein in Escherichia coli.

We describe the results of a study by electron microscopy and image processing of Gag protein shells-immature capsids--of Mason-Pfizer monkey virus assembled in Escherichia coli from two truncated forms of the Gag precursor: Deltap4Gag, in which the C-terminal p4Gag was deleted, and Pro(-)CA.NC, in which the N-terminal peptides and proline 1 of the CA domain were deleted. Negative staining of capsids revealed small patches of holes forming a trigonal or hexagonal pattern most clearly visible on occasional tubular forms. The center-to-center spacing of holes in the network was 7.1 nm in Deltap4Gag capsids and 7.4 nm in Pro(-)CA.NC capsids. Image processing of Deltap4Gag tubes revealed a hexagonal network of holes formed by six subunits with a single subunit shared between rings. This organization suggests that the six subunits are contributed by three trimers of the truncated Gag precursor. Similar molecular organization was observed in negatively stained Pro(-)CA.NC capsids. Shadowed replicas of freeze-etched capsids produced by either construct confirmed the presence of a hexagonal network of holes with a similar center-to-center spacing. We conclude that the basic building block of the cage-like network is a trimer of the Deltap4Gag or Pro(-)CA.NC domains. In addition, our results point to a key role of structurally constrained CA domain in the trimeric interaction of the Gag polyprotein.

Localization of the Linker Domain of Ca2+/Calmodulin-Dependent Protein Kinase II

Electron micrographs of rotary shadowed replicas of α-Ca2+/calmodulin-dependent protein kinase II reveal a flower-shaped multimeric molecule with a central particle surrounded by 8–10 smaller peripheral particles. Peripheral particles are attached to the central particle by thin arms or “linkers.” Movement of peripheral particles to contact each other for autophosphorylation is likely to involve these linkers. It has generally been accepted that the segment 317–328 of the α-subunit constitutes the linker domain. In the present study we test this assumption by generating a mutant lacking the proposed sequence. The mutant has biochemical and morphological properties similar to those of the wild type, and a thin linker is occasionally observed in replicas from either type. The results indicate that the deleted sequence does not correspond to the linker domain. This conclusion, combined with observations from two recent studies which identify the C-terminal domain involved in oligomerization, narrows down the location of the linker domain within the sequence 330–354.

Ultrastructural modifications of the extracellular matrix upon calcification of growth plate cartilage as revealed by quick-freeze deep etching technique.

The ultrastructural changes in cartilage matrix that occur during calcification have been examined in chick epiphyseal growth plate cartilage prepared by quick-frozen, deep-etched, and rotary shadowed replicas. The extracellular cartilage matrix contains a reticular network closely associated with an extensive network of collagen. The components of the reticular network, including thick and thin filaments, are attached directly to the cell membrane, matrix vesicle membrane, and collagen fibrils. This network, which interconnects the matrix vesicles and collagen, fills the extracellular matrix. The dimensions of the reticular network seem to remain almost constant in size from the reserve and proliferative zones to the calcifying zone. The collagen fibrils seem to consist of subfibrillar structures that branch and anastomose. In optimally quick-frozen, deep-etched, prepared collagen, a cross-banding pattern was exposed. Globular structures stud the collagen fibrils, which gradually diminish in number from the reserve zone down to the calcifying zone. The matrix vesicles, when fractured, showed a granular appearance. In most cases, the fracture plane passed through the bilayer of the matrix vesicle membrane. The true surface of the matrix vesicle membrane, therefore, was exposed after deep etching. At the calcifying zone, crystal deposition had occurred in needle-like and/or plate-like form within the membrane-bound matrix vesicles. The reticular network was still intact in the vicinity of the calcified matrix, but in the intercrystalline space, neither the reticular structure nor the globular structure was detectable. Within the calcified matrix, both reticular and granular structures had disappeared from the interfibrillar space of the collagen fibrils.

The loci of mineral in turkey leg tendon as seen by atomic force microscope and electron microscopy

Transmission electron micrographs of fully mineralized turkey leg tendon in cross-section show the ultrastructure to be more complex than has been previously described. The mineral is divided into two regions. Needlelike-appearing crystallites fill the extrafibrillar volume whereas only platelike crystallites are found within the fibrils. When the specimen is tilted through a large angle, some of the needlelike-appearing crystallites are replaced by platelets, suggesting that the needlelike crystallites are platelets viewed on edge. If so, these platelets have their broad face roughly parallel to the fibril surface and thereby the fibril axis, where the intrafibrillar platelets are steeply inclined to the fibril axis. The projection of the intrafibrillar platelets is perpendicular to the fibril axis. The extrafibrillar volume is at least 60% of the total, the fibrils occupying 40%. More of the mineral appears to be extrafibrillar than within the fibrils. Micrographs of the mineralized tendon in thickness show both needlelike-appearing and platelet crystallites. Stereoscopic views show that the needlelike-appearing crystallites do not have a preferred orientation. From the two-dimensional Fourier transform of a selected area of the cross-sectional image, the platelike crystallites have an average dimension of 58 nm. The needlelike-appearing crystallites have an average thickness of 7 nm. The maximum length is at least 90 nm. Atomic force microscopy (AFM) of unstained, unmineralized turkey leg tendon shows collagen fibrils very much like shadow replicas of collagen in electron micrographs. AFM images of the mineralized tendon show only an occasional fibril. Mineral crystallites are not visible. Because the collagen is within the fibrils, the extrafibrillar mineral must be embedded in noncollagenous organic matter. When the tissue is demineralized, the collagen fibrils are exposed. The structure as revealed by the two modalities is a composite material in which each component is itself a composite. Determination of the properties of the mineralized tendon from the properties of its elements is more difficult than considering the tendon to be just mineral-filled collagen.

Further Evidence of Icosahedral Symmetry in Human and Simian Immunodeficiency Virus

Specimens of HIV and SIV have been examined by electron microscopy, using the techniques of conventional thin sectioning, freeze-substitution, cryosectioning, and cryomicroscopy of frozen hydrated specimens. In addition freeze-drying and critical point drying were used for both shadowed replicas and scanning electron microscopy. Thin sections revealed hexagonal, pentagonal, or near-spherical profiles. Angular particles were seen in shadowed replicas and also by scanning electron microscopy. The images observed were consistent with an icosahedral shape of the virus. It is proposed that mature HIV (SIV) is an icosadeltahedron with flat triangular facets. Size measurements of the specimens showed a wide range of values for conventional embedding, but a narrow range for specimens prepared by low-temperature techniques.

Distribution of avian integrin during the lifetime of chicken embryo fibroblasts in vitro: Study by immunofluorescence and immuno electron microscopy

The localization of avian integrin has been studied in chicken embryo fibroblasts (CEF) with antibodies to both the external and cytoplasmic domains of the β 1 subunit and using a variety of immunolabelling methods at the light and electron microscopy level. Cells were studied during the spreading, the locomotory and the stationary phase of the culture. Spreading cells adhered via typical peripheral focal contacts and also through small ‘point-contacts’. Both types of adhesions contained the β 1 integrin. Cells from a subconfluent culture consisting mainly of motile cells revealed only occasionally focal contacts (FC), more frequently ‘point-contacts’ (PC) or extracellular matrix contacts (EMC). Stereo micrographs of whole mount preparations as well as thin sections of cells showed about equal density of receptor on both cell surfaces (dorsal and ventral). Frequently the receptors appeared in clusters. High density of receptors was seen at the cell edges. Focal and extracellular matrix contacts were frequent in stationary cells, but only about half of them contained the β 1 integrin. The presence of integrin in cell/substratum adhesions was demonstrated not only by immunofluorescence microscopy, but also by electron microscopy using protein A/horseradish peroxidase procedure in situ followed by thin sectioning, also in shadowed replicas of both the dorsal and ventral membrane surfaces immunolabelled with colloidal gold. However, gold immunolabelling did not reveal the receptor when carried out on monolayers ( in situ) suggesting that the colloidal gold conjugated antibody cannot penetrate the narrow gap between the cell and the substratum. Replicas of gold immunolabelled protoplasmic surfaces of ventral membranes revealed a close contact between the receptor and extracellular fibronectin cables.

How is the fractal dimension of a thin film top surface connected with the roughness parameters and anisotropy of this surface?

It was previously shown how the fractal dimension of thin films top surfaces can be obtained from the surface relief reconstructed by microdensitometer analysis of an electron micrograph of a shadowed replica of the surface. Here is presented a comparative study of roughness, fractal dimension and anisotropy of thin films top surfaces. After a theoretical study devoted to some regular and more or less isotropic surfaces, results are given for several real surfaces and it is shown how the characterization of a rough surface by its fractal dimension yields results about its roughness and anisotropy.

Isolation and localization of a spectrin-like protein from echinoderm sperm

Thyone sperm undergo an explosive acrosome reaction resulting in the extension of a 90 microns long acrosomal process. In unreacted sperm, profilamentous actin is sequestered within the profilactin cup (Tilney: Journal of Cell Biology 69:73-89 1976), which consists of four major polypeptides: actin, profilin, and a 250/235 kDa equimolar doublet (TS 250/235). Dialysis of profilactin preparations into an actin assembly buffer resulted in the formation of acrosomal-like macromolecular aggregates containing actin, TS 250/235, and several other polypeptides as detected by SDS-PAGE. TS 250/235 was purified by subjecting extracts of pH solubilized profilactin cups to DEAE and phosphocellulose ion exchange chromatography. TS 250/235 demonstrated immunocrossreactivity with affinity purified polyclonal antibodies raised against S. purpuratus egg spectrin. As determined by biotinylated-calmodulin overlays, both subunits of TS 250/235 bound calmodulin in a Ca(++)-sensitive manner. Electron microscopy of low angle, rotary shadowed replicas of TS 250/235 revealed an elongate rod-shaped molecule with an average contour length of 203 nm. By indirect immunofluorescence, TS 250/235 was found to be uniformly distributed throughout the profilactin cup of the unreacted sperm. This distribution of TS 250/235 correlated with the location of monomeric actin as determined by localization studies utilizing fluorescent-DNase-1. Upon sperm activation, the cellular distribution of TS 250/235 dramatically changed and was observed both along the length and at the base of the extended acrosomal process.

Direct visualization of cross-links in the primary plant cell wall

ABSTRACT We have investigated the structure of the onion primary cell wall at high resolution, using shadowed replicas of rapidly frozen deep-etched specimens. We have sequentially extracted polymers from the wall and have visualized both these and the remaining structures at each extraction step. By viewing the structures in as near their native state as possible, an accurate three-dimensional picture of wall construction has been assembled, facilitated by viewing stereo pairs of micrographs. Our observations show that the physical links between cellulose microfibrils that we observe in the intact wall are generally shorter (20-40 nm) than the isolated molecules we extract (30->700nm), suggesting that lateral interactions must occur between linking polymers and cellulose in muro. These cross-links are hemicellulosic and we believe them to be xyloglucans: their removal allows increased lateral association of microfibrils. Na2CO3-extractable pectic fractions form a separate coextensive network, the removal of which does not affect basic cellulose/ hemicellulose architecture. Preliminary evidence for a lamellate model of wall construction has been obtained. In addition, we propose a positive role for hemicellulose in maintaining the ordered spacing of cellulose microfibrils, perhaps regulating wall porosity and strength. The basic wall parameters that we derive impose constraints on possible cell wall models.

Improvement in the method of searching for the fractal nature of rough thin-film top surfaces

It was previously shown how the fractal nature of thin-film top surfaces can be obtained from the surface relief reconstructed by microdensitometer analysis of an electron micrograph of a shadowed replica of the surface. Here, we present some improvement in the determination of the fractal dimension Dl which characterizes a planar section of the thin film, as well as a different way of conducting such a determination. Results connected with several rough thin films are given and it is found that many different ways of characterizing the fractal nature of a thin-film top surface are possible from the reconstructed relief of the surface to be studied.

Defining the fractal nature of thin‐film top surfaces from microdensitometer analysis of electron micrographs of surface replicas

In this paper, the very first results are reported about searching for the fractal nature of thin‐film top surfaces from the surface relief reconstructed by microdensitometer analysis of an electron micrograph of a shadowed replica of the surface. It is found that such a reconstruction could be very helpful when dealing with the fractal nature of a surface.

Quantitation of intramembrane particles of Mcleod red cells

The McLeod phenotype is characterized by numerous acanthocytic red cells. Glaubensklee et al report a significant increase in numerical density and decrease in the volume of intramembrane particles (IMP) of McLeod red cells on unidirectionally shadowed replicas of freeze fractured membranes and suggest that the dissociation of band 3 glycoprotein in this membrane may underlie the abnormal shape. Rotary shadowing has several advantages over unidirectional shadowing rendering it more reliable for IMP counts. In this study we compare and quantitate the numerical densities of the IMP of red cells of normal donor and McLeod patient on rotary shadowed replicas.

Ultrastructure and chemical composition of the ballistospore wall of Conidiobolus obscurus

The ballistospores of the entomopathogen Conidiobolus obscurus are spheroidal cells with a papilla corresponding to the zone of attachment on the sporophore. They are covered by a mucus responsible for the attachment of the spore to the insect. Chemical and cytochemical investigations of the nature of the wall components have been undertaken to better understand fungus-insect interactions in entomopathology. β(1→3)-Glucans and chitin together represented the main components of the wall which did not contain chitosan and uronic acids. Transmission electron microscopy revealed that the spore wall was composed of a thick electron-lucent inner layer and a thin outer electron-dense layer, the latter being absent at the papilla region. The spore is covered by a mucilaginous layer that upon spore impact on a substratum, is dispersed and forms a halo around the spore. Shadow replicas of the discharged spores showed that they are covered by rodlets except on the papilla which displayed a chitinous, microfibrillar structure. The ontogeny of the rodlets deposited on the surface of young spores was characterized by a progressive organization of separate rodlets and then a clustering of the rodlets in fascicles. Shadow replicas and chemical and enzymatic investigations of the halo surrounding discharged spores showed that the mucus was composed of long β(1→3)-glucan microfibrils embedded in amorphous proteins partly covered by rodlets discharged from the spore surface.

Microdensitometer analysis of electron micrographs of shadowed surface replicas: A powerful method in surface structure characterization

Microdensitometer analysis of electron micrographs of shadowed surface replicas: A powerful method in surface structure characterization

Influence of some parameters on the surface profile restored from microdensitometer analysis of electron micrographs of surface replicas

From the micrograph density of a shadowed carbon replica of a surface it is possible, by using a microdensitometer analysis, to rebuild line by line the profile of that surface. After summarizing the different stages necessary for such a reconstruction, we study the influence on the restored profiles of the various parameters that are encountered during the various steps of experiment and computation. It is shown that the most important parameters are the shadow-casting angle, microdensitometer scanning aperture, and filtering characteristics. Other parameters such as magnification micrograph, microdensitometer scanning speed, and microdensitometer noise play a minor role. The best way to choose and fit the appropriate value of the various parameters is discussed.

Imaging of platinum-shadowed macromolecular complexes in dark field

Shadowing with heavy metals has been used for many years to enhance the topological features of biological macromolecular complexes. The three dimensional features present in directionaly shadowed specimens often simplifies interpretation of projection images provided by other techniques. One difficulty with the method is the relatively large amount of metal used to achieve sufficient contrast in bright field images. Thick shadow films are undesirable because they decrease resolution due to an increased tendency for microcrystalline aggregates to form, because decoration artefacts become more severe and increased cap thickness makes estimation of dimensions more uncertain.The large increase in contrast provided by the dark field mode of imaging allows the use of shadow replicas with a much lower average mass thickness. To form the images in Fig. 1, latex spheres of 0.087 μ average diameter were unidirectionally shadowed with platinum carbon (Pt-C) and a thin film of carbon was indirectly evaporated on the specimen as a support.

Improvement in the reconstruction of surface profile deduced from microdensitometer analysis of electron micrographs of shadowed surface replicas

The different steps leading to the determination of a surface profile deduced from microdensitometer analysis of electron micrographs of shadowed surface replicas are reviewed. It is shown that monodimensional median filtering is sometimes inadequate to remove the low-frequency noise generated by the Levy–Wiener process. A new treatment is proposed that corrects this disadvantage.

Spectral-density function of the surface roughness for polished optical surfaces

The spectral density functions g(k) of the surface roughness for polished optical surfaces of CaF2, MgF2, and LiF are computed from autocovariance functions, which in turn have been determined from surface profiles obtained by using a microdensitometer analysis of electron micrographs of shadowed surface replicas. The fast-Fourier-transform algorithm and a smoothing procedure have been used to determine the g(k) estimates. It is shown that g(k) is not a Gaussian function throughout, as is usually assumed. Spectral density functions gS′(k) of the surface slopes are also computed, and it is shown that results obtained are consistent with those deduced from g(k). Limitations of our method at low spatial frequency are discussed. An analytical model for g(k) is investigated that should be useful in performing theoretical calculations on scattering problems.