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PubMedDate not supplied

[Isolation of erythroblastic islands. Study by optical and scanning electron microscopy (author's transl)].

1. A simple technique is described for isolation and estimation of number of erythroblastic islands (EBI) in rat bone marrow. The number of EBI decreases during hypertransfusion polycythemia, and increases during experimentally-induced hemolytic anemia. It has also been possible to maintain EBI in vitro, under culture conditions, for up to 36 hours. 2. Each EBI contains a central cell having the character of a macrophage-histiocyte which adheres to glass and spreads, while still retaining its connections with the erythroblasts. 3. In an EBI the erythroblasts are arranged in the form of concentric rings, with the outer cells being more mature than the inner. The outermost layer is frequently made up of reticulocytes even in the isolated island. The erythroblasts of a single ring appear to be at the same stage of maturation which suggests that they are derived from the same stem cell. 4. Using a coordinate reference system, studies of nuclear expulsion were made, by both optical microscopy and scanning electron microscopy.

Animals↗
PubMed1978

Cell surface changes in capping studied by correlated fluorescence and scanning electron microscopy.

A simple method has been devised for correlating fluorescence microscopy and scanning electron microscopy, whereby the identical cell observed by the former can be observed by the latter. With this methodology, we have studied the sequence of cell surface changes which occur when mouse B lymphocytes, bearing immunoglobulin (Ig) on their surfaces, interact with fluoresceinated anti-Ig antibodies. Initially, the pattern of staining is diffuse; then, rapidly, patching occurs, followed by the sweeping of the patches into a cap. Accompanying these events are: the disappearance of microvilli, the formation of ruffles and lamellipodia at the pole of the cell opposite the capped pole, the presence of a constriction ring beneath the cap, ameboid shapes, and the translocation of the cell in a direction opposite the cap. Whereas the cell body is smooth, the capped pole frequently shows the presence of numerous microvilli. However, not all capping cells show the changes, and cells capped in the presence of cytochalasin B usually show none of them, except that their surfaces are smooth. Furthermore, T-cells undergoing translocation show the same cell surface changes as do B-cells. Whereas the contractile apparatus of the cell is considered to underlie all these phenomena, it is concluded that the cell surface changes do not result from patching and capping per se, but rather are an expression of the cell translocation also induced by the anti-Ig antibody.

Animals↗
PubMedDate not supplied

Hot isostatic pressing-processed hydroxyapatite-coated titanium implants: light microscopic and scanning electron microscopy investigations.

Hot isostatic pressing (HIP) was used in a new procedure to produce hydroxyapatite (HA) coatings on a commercially pure titanium (cpTi) substrate for osseous implantation. Eighteen HIP-processed HA-coated implants were placed in the inferior border of the mandibles in 2 Labrador retriever dogs and left submerged for 3 months. As control specimens, 12 sandblasted cpTi implants were placed in the same mandibles and, to compare the bone reaction, 2 additional plasma-sprayed HA-coated implants (Integral) were placed. Tissue reactions at the bony interfaces of the implants were studied in ground sections with the implants in situ, using ordinary, fluorescent, and polarized light microscopy and scanning electron microscopy (SEM). The HIP-processed HA coatings displayed an increased density in light microscopy and SEM as compared to plasma-sprayed coatings. Direct bone-implant contact was found in all 3 types of surfaces. However, the production of new bone was far more abundant for the HA-coated implants than for sandblasted cpTi implants. The presence of bone-forming and bone-resorbing cells indicated active bone remodeling in the interface area at 3 months after implant placement. The present results support the view that epitaxial bone growth may occur from the HA-coated implant surface. It was concluded that the increased density of the present HIP-processed HA material does not reduce the bioactive properties of the coatings.

Animals↗
PubMed2001

Surface ultrastructure of collagen fibrils and their association with proteoglycans in human cornea and sclera by atomic force microscopy and energy-filtering transmission electron microscopy.

PURPOSE: We aimed to investigate the possible association of proteoglycans with D-periodic collagen fibrils in the human cornea and sclera, using energy-filtering transmission electron microscopy (EF-TEM) and atomic force microscopy (AFM). METHODS: Human cornea and sclera were digested with keratanase to eliminate keratan sulfate proteoglycans (KSPGs). For EF-TEM observation, surface proteoglycans were detected by cupromeronic blue (CB) staining. For AFM observation, cornea and sclera were treated with sodium hydroxide before and after keratanase digestion, and the surface topology of collagen fibrils was analyzed. RESULTS: With CB staining, numerous CB-positive short filaments of surface proteoglycans (proteoglycan filaments) were observed in the interfibrillar spaces of cornea and sclera associated with collagen fibrils. AFM imaging showed that the depth and periodicity of D-periodic collagen fibrils in keratanase-treated corneal collagens were deeper and more regular than in untreated ones. Moreover, the depth and periodicity of keratanase-untreated corneal collagens were shallow and irregular in comparison with keratanase-untreated scleral collagens. On the other hand, there was no difference in depth or regularity between keratanase-treated and -untreated scleral collagen fibrils. Using AFM imaging, additional thin grooves sub-bands were detected on the surface of keratanase-treated corneal collagen fibrils. The grooves were not detected in keratanase-untreated collagen fibrils nor in scleral collagen fibrils with or without keratanase digestion. Comparing densitometry waves, the grooves of D-periodic corneal collagen sub-bands corresponded to a and c bands. CONCLUSION: Using AFM and EF-TEM to study corneal and scleral collagen fibrils and their association with proteoglycans, we conclude that KSPG is found in ample amounts in the human cornea in comparison with sclera. Moreover, we topologically detected KSPG attached to a and c bands of collagen fibrils.

Aged↗
PubMed1979

Morphologic effects of pressure changes on canine carotid artery endothelium as observed by scanning electron microscopy.

By using scanning electron microscopy (SEM) we were able to follow the sequence of ultrastructural alterations of the luminal surface which occurred when specimens of canine carotid artery were subjected to controlled transmural pressures. Specimens of carotid artery were removed from dogs following fixation at experimental pressures ranging from 0 mm Hg to 100 mm Hg. The endothelium of specimens fixed at 0 mm Hg has parallel longitudinal ridges formed by the contraction of the underlying internal elastic lamina. With increasing transmural pressure, the luminal surface undergoes a gradual flattening of the endothelial ridges so that at 100 mm Hg, these ridges have completely disappeared. The observed morphologic changes of the arterial endothelium indicate that SEM can provide good ultrastructural information on blood vessels subjected to controlled transmural pressure and that the pressure-dependent alterations must be considered in studies on vascular structure and function.

Animals↗
PubMed1990

Three serotypes of Norwalk-like virus demonstrated by solid-phase immune electron microscopy.

Solid phase immune electron microscopy (SPIEM) was used to investigate the serological differences between Norwalk-like virus (NLV) strains from five different outbreaks within the United Kingdom. The existence of two previously demonstrated serotypes, Lewis et al. (Journal of Clinical Microbiology 26:938-942, 1988), was confirmed by the use of whole convalescent sera and purified IgM. A third serotype was found to be the agent of two recent hospital outbreaks and could similarly be typed by use of whole sera or pure IgM. Paired sera were available for two of the three serotypes and demonstrated rising antibody levels. These antibody rises were also specific for the infecting serotype. However, two serum pairs from a later outbreak gave antibody rises to all three serotypes, although much higher counts were produced with the infecting serotype. SPIEM is a useful method for distinguishing NLV serotypes and can also be used to detect specific IgM and to demonstrate seroconversion. Cross-reacting antibodies, possibly anamnestic in origin, can occur after natural infection and could cause confusion in typing virus unless further evidence of the identity of the infecting agent is obtained.

Adult↗
PubMed1975

Electron microscopic studies of the lung of the frog. II. Topography of the inner surface by scanning and transmission electron microscopy.

Scanning and transmission electron microscopy were used to study the inner architecture of the frog lung. In some specimens the alveolar surface mucus layer was removed to permit the examination of underlying features. The inner surface of the frog's lung is covered by a layer of microvilli belonging to only one type of epithelial cells. The boundaries of these epithelial cells are demarcated by small ridges. Different degrees of lung expansion cause variations of the surface topography. The morphology of certain surface features is examined in detail. Several methods of drying the specimens are compared.

Animals↗
PubMed1987

Detection of protein-free lipoprotein analogues with an apolar lipid core by freeze-etch electron microscopy.

Freeze-fracture electron microscopy of protein-free lipoprotein analogues consisting of polar phospholipids with neutral lipid cores shows that these systems can exhibit novel, smooth crossfracture faces. Resolution of these features can be enhanced by etching techniques. This novel particle morphology provides a new procedure to characterize microemulsions composed of polar and neutral lipids.

Emulsions↗
PubMed1989

Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy.

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.

Adenosine Triphosphate↗
PubMed1990

Studies of crystal growth mechanisms of proteins by electron microscopy.

We have used electron microscopy to examine the surfaces of lysozyme crystals and deduce mechanisms of crystal growth. We find that growth occurs by a lattice defect mechanism at low supersaturation and by two-dimensional nucleation at high supersaturation. Step velocities and two-dimensional nucleation rates are obtained, and their dependence on supersaturation is compared with theory. Some features of the observed surface structure can be related to the specific topology and strengths of the bonds in the P4(3)2(1)2 lattice. Preliminary results on the early stages of nucleation and the phenomenon of cessation of growth are presented.

Crystallization↗
PubMed1980

A new phenomenon (SMOG) associated with solid phase immune electron microscopy.

Solid phase immune electron microscopy (SPIEM) has been used to study the interaction of virus and antibody both quickly and with economy of reagents. Unexpectedly, the present study shows that when virus-coated grids are floated on drops of specific antibody the virus particles migrate to form complexes. This has been termed specific migration on the grid (SMOG) and it is suggested that it can be used both to assess the properties of an antiserum and to make virus more readily detectable. The mechanism by which SMOG occurs is not understood but various possibilities are discussed.

Antibodies, Viral↗
PubMed2001

Ultrastructural morphology and localisation of cisplatin-induced platinum-DNA adducts in a cisplatin-sensitive and -resistant human small cell lung cancer cell line using electron microscopy.

Ultrastructural morphology (transmission electron microscopy) and localisation of cisplatin-induced platinum (Pt)-DNA adducts (immunoelectron microscopy) were analysed in the human small cell lung cancer cell line GLC(4) and its 40-fold in vitro acquired cisplatin-resistant subline GLC(4)-CDDP, which is characterised by, among other things, a decreased DNA platination. Immunolabelling of Pt-DNA adducts was performed with the polyclonal antibody GPt, known to detect the main Pt-containing intrastrand and interstrand DNA adducts. Morphological analysis of GLC(4) and GLC(4)-CDDP at the ultrastructural level showed cells with a high nucleus/cytoplasm ratio with the majority of nuclei containing one or more nucleoli. GLC(4)-CDDP showed, in contrast to GLC(4), an extensive Golgi apparatus and an increased number of mitochondria. DNA platination was detectable in both GLC(4) and GLC(4)-CDDP. Immunoelectron microscopy showed Pt-DNA adducts primarily in the nucleus, preferentially at loci with high-density chromatin (e.g. heterochromatin, pars granulosa around nucleoli, condensed DNA in proliferating and apoptotic cells), and in mitochondria. The level of detectable Pt-DNA adducts was cell cycle status-dependent. In both cell lines, Pt-DNA adduct levels increased from non-dividing interphase cells to dividing cells and were highest in cells undergoing apoptosis. Overall localisation of Pt-DNA adducts was comparable in GLC(4) and GLC(4)-CDDP cells.

Antibodies↗
PubMed2003

Distribution of the attachment (G) glycoprotein and GM1 within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy.

Field emission scanning electron microscopy (FE SEM) was used to visualize the distribution of virus-associated components, the virus-attachment (G) protein, and the host-cell-derived lipid, GM1, in respiratory syncytial virus (RSV) filaments. RSV-infected cells were labeled in situ with a G protein antibody (MAb30) whose presence was detected using a second antibody conjugated to colloidal gold. No bound MAb30 was detected in mock-infected cells, whereas significant quantities bound to viral filaments revealing G protein clusters throughout the filaments. GM1 was detected using cholera toxin B subunit conjugated to colloidal gold. Mock-infected cells revealed numerous GM1 clusters on the cell surface. In RSV-infected cells, these gold clusters were detected on the filaments in low, but significant, amounts, indicating the incorporation of GM1 within the viral envelope. This report describes the first use of FE SEM to map the distribution of specific structural components within the envelope of a Paramyxovirus.

Animals↗
PubMed1981

Distribution of receptor sites on large glycoprotein molecules by electron microscopy. Application to epiglycanin.

Electron microscopy was used to map the loci of immunochemically active sites on individual glycoprotein molecules. The positions of specific galactose residues and asparagine-linked carbohydrate chains containing specific mannose residues in epiglycanin, a glycoprotein of extended conformation from the surface of TA3 mouse mammary tumour cells, were observed in complexes with Ricinus communis toxin and concanavalin A respectively. The maximum number of Ricinus communis toxin molecules attached to a single epiglycanin molecule was 23, and the average number was 16. Only one concanavalin A molecule was observed attached to any epiglycanin molecule, and this at one end of the molecule, suggesting the presence of only one receptor for this lectin. By means of this new approach for mapping specific residues, evidence has been obtained that suggests microheterogeneity in epiglycanin with respect to the locations of carbohydrate chains containing receptors for Ricinus communis toxin.

Animals↗
PubMed1974

Use of the avidin-biotin complex for specific staining of biological membranes in electron microscopy.

To expand the electron microscopist's options in localization and visualization, a new and general staining technique has been tested. The avidin-biotin complex serves as a coupling between the electron-dense marker, ferritin, and points of interest in biological samples. When specific cellular components are tagged with biotin, those components may be visualized with ferritin-linked avidin. Because of the remarkably strong affinity of avidin and biotin (characterized by an association constant of 10(15) M(-1)), the staining is rapid and stable. The preparation of ferritin-avidin conjugate is described, and examples are presented of the application of this complex to biotin-tagged membranes. The ghosts of Acholeplasma laidlawii have been treated with biotinyl-N-hydroxysuccinimide ester to label protein amino groups. Erythrocyte membrane oligosaccharides have been oxidized by periodate or by galactose oxidase, and the resulting aldehydes labeled with biotin hydrazide. The avidin-biotin complex in electron microscopy seems especially appropriate for seqential staining procedures, as well as for visualization of reaction sites of biotin-labeled, low-molecular-weight reagents.

Acholeplasma laidlawii↗
PubMed1975

Chromatin architecture: investigation of a subunit of chromatin by dark field electron microscopy.

Dark field scanning electron microscopy of unstained, unfixed samples of chromatin, histone-1-depleted chromatin, and nucleohistone has been used to identify an apparent subunit of chromatin, namely a disk-shaped structure we term the unit particle, which is probably about 135 A wide and 50 A thick in the hydrated state. The unit particles are found at rather uniform intervals along thin DNA-like fibers. Histone 1 depletion leads to a bimodal distribution of these spacings. Our observations suggest that the unit particle consists of a loop of nucleoprotein, perhaps around a histone core.

Animals↗
PubMed1986

Differentiation of infective larvae of Brugia malayi and Wuchereria bancrofti by scanning electron microscopy.

A comparative scanning electron microscopy of the infective (third stage) larva of Wuchereria bancrofti and Brugia malayi revealed the following features: The lateral caudal papillae in both species are rounded and the terminal papilla is relatively pointed and smaller. The caudal papillae in W. bancrofti are more protruding while in B. malayi they are flattened. The lateral caudal papillae in B. malayi have an indentation around their bases which is absent in W. bancrofti. This is the most useful distinguishing feature and easily recognizable. The oral opening and the cephalic papillae in both the species are similar and not useful for differentiation.

Animals↗
PubMed1982

Effects of amphotericin B and its methyl ester on plasma membranes of Candida albicans and erythrocytes as examined by freeze-fracture electron microscopy.

Freeze-fracture electron microscopy of the plasma membranes of Candida albicans yeast cells and red blood cells treated with amphotericin methyl ester and amphotericin B showed that amphotericin B (50 micrograms ml-1) caused extreme aggregation of intramembranous particles on the protoplasmic fracture face of the C. albicans membrane, and a marked reduction of the density of intramembranous particles. On the other hand, the rearrangement of intramembranous particles induced by amphotericin methyl ester (50 micrograms ml-1) produced elevations of the particle-free membrane domains toward the outside of the cells, so that the particles were aggregated in linear furrows surrounding these elevations on the protoplasmic fracture face, and the corresponding ridges on the exoplasmic fracture face. The density of intramembranous particles was greatly reduced on the protoplasmic fracture face. Both polyenes produced only small changes in the erythrocyte membranes at the same concentration. These results suggest that amphotericin methyl ester affects the ergosterol-containing membranes more than amphotericin B, and that ergosterol has a higher sensitivity for these two polyene antibiotics than cholesterol.

Amphotericin B↗