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At least 19 recordsLinked to original sources
PubMedDate not supplied

Visualization of a ternary complex of the Escherichia coli Phe-tRNA(Phe) and Tu.GTP from Thermus thermophilus by scanning transmission electron microscopy.

Scanning transmission electron microscopy (STEM) was used to visualize formation of a ternary complex between the T. thermophilus elongation factor (EF) Tu.GTP and the Escherichia coli Phe-tRNA(Phe) labeled with an undecagold (Au11) cluster at minor nucleotide 3-(3-amino-3-carboxypropyl) uridine at position 47. The ternary complex was further characterized by the molecular mass and radius of gyration calculated from the mass distribution within the individual particles. Under conditions used for STEM imaging, the ternary complex is formed between Au11-labeled Phe-tRNA(Phe) and Tu.GTP in a yield up to 25%. The stoichiometry of EF-Tu.GTP to aminoacyl-tRNA (aa-tRNA) in the EF-Tu.GTP.aa-tRNA complex is 1:1, in agreement with the established view of the protein biosynthesis mechanism. The ternary complex is also formed, although to a lower extent, with GTP analogues (GMPPCP and GMPPNP, respectively), but not with Tu.GDP and nonaminoacylated tRNA(Phe) with Tu.GTP.

Escherichia coli↗
PubMed1997

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy.

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin metal coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the "flower-like" structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.

Adrenal Cortex↗
PubMedDate not supplied

Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy.

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the 80S monosomes and the large (60S) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the "native" eukaryotic ribosome 3-D model.

Animals↗
PubMed1981

Improved contrast in cytochemistry of dehydrogenases by scanning transmission electron microscopy.

A scanning transmission electron microscope (STEM) was used to examine ultrathin sections of rabbit white skeletal muscle. Lactic dehydrogenase (LDH) activity was localized in the tissue using the tetra-nitro blue tetrazolium (TNBT) method. For most specimens postfixation was omitted in order to avoid reoxidation and solubilization of the formazan by osmium tetroxide. The STEM image revealed sufficient contrast of the intracellular structures and apparently electron-dense reaction product in the sarcoplasmic reticulum and mitochondria. Substantially less contrast was obtained when the same areas were observed by conventional transmission electron microscopy (CTEM). In material postfixed with osmium tetroxide, although the tissue contrast was improved, the TNBT reaction product was focally leached out, exhibiting lower contrast than in unosmicated sections. These results indicate that the fine structural visualization of dehydrogenases with TNBT, the STEM technique as used in the present study is superior to that obtained by CTEM.

Animals↗
PubMed1978

Molecular weight determination by scanning transmission electron microscopy.

A scanning transmission electron microscope is employed to determine the mass of biological macromolecules. Elastically scattered electrons are collected by an annular detector that is capable of counting single electrons. Off-line processing of these dark field micrographs stored on a magnetic tape is accomplished by a mini-computer. It allows the number of electrons scattered by spherical or filamentous proteins to be evaluated. The calibration factor relating the number of scattered electrons to the mass of the protein is derived from scattering theory and is experimentally determined from biological macromolecules of known mass. Mass-loss kinetics of biological specimens due to the electron beam are measured for various protein structures. The application of this method is illustrated by determination of the mass of an oligometric protein (major phage T4 head protein) and the mass per unit length of a filamentous protein aggregate (F-pili). The unique possibilities of this new technique as well as its limitations are discussed.

Bacterial Proteins↗
PubMed1977

The rat epithalamus. I. Correlative scanning-transmission electron microscopy of supraependymal nerves.

Transmission and scanning electron microscopy of the rat epithalamus shows a regional variation in the distribution of supraependymal nerves (SN) which correlates well with supraependymal yellow fluorescence reported by Richards et al. (1974). The medial habenular nucleus, the intercommissural and suprahabenular recesses, the habenular commissure and the fibrae periventriculares thalami have the greatest density of SN/100micron of ependymal surface. The floor of the suprahabenular and intercommisural recesses is covered by non-ciliated ependyma. The significance of these findings is discussed with respect to (1) a direct functional relationship of SN with ependyma, and (2) a possible participation of the non-ciliated ependyma of the suprahabenular and intercommissural recesses in secretory activity whereby the CSF serves as a vehicle for neuroendocrine communication.

Animals↗
PubMed1996

A study of Technegas employing X-ray photoelectron spectroscopy, scanning transmission electron microscopy and wet-chemical methods.

Scanning transmission electron microscopy (STEM), coupled with energy dispersive X-ray analysis (EDS), X-ray photoelectron spectroscopy (XPS) or radionuclear chemical methods, indicates that the active agent in Technegas is either polymeric TcO2[i.e. (TcO2)n] or (TcO2)n bound to a carbon nanoparticle. The particle size observed using STEM is in good agreement with other published results. XPS has also been used to investigate technetium residues remaining on spent crucibles. The chemical form of technetium in this residue is quite different to the form detected in the aerosol particles. We conclude that the small fraction that migrates into the crucible framework upon resistive heating is reduced to either metallic technetium or carbidic forms, with the remaining nuclide evaporating as (TcO2)n with or without carbon before complete reduction can occur.

Chemistry↗
PubMed1993

Application of scanning transmission electron microscopy to the study of biological structure.

The scanning transmission electron microscope provides structural and chemical information of a specimen at atomic-scale resolution and complements conventional transmission electron microscopy techniques. Mass measurements can now be performed routinely on a wide range of molecular and supramolecular structures using elastically scattered electrons. Recent progress in the acquisition and analysis of electron energy-loss spectroscopy data indicates that the scanning transmission electron microscope is an efficient tool for mapping the chemical composition of biological samples.

Biotechnology↗
PubMed1994

Scanning transmission electron microscopy of biological structures.

The design of the scanning transmission electron microscope (STEM) has been conceived to optimize its detection efficiency of the different elastic and inelastic signals resulting from the interaction of the high energy primary electrons with the specimen. Its potential use to visualize and measure biological objects was recognized from the first studies by Crewe and coworkers in the seventies. Later the real applications have not followed the initial hopes. The purpose of the present paper is to describe how the instrument has practically evolved and recently begun to demonstrate all its potentialities for quantitative electron microscopy of a wide range of biological specimens, from freeze-dried isolated macromolecules to unstained cryosections. Emphasis will be put on the mass-mapping, multi-signal and elemental mapping modes which are unique features of the STEM instruments.

Bacteriophage T4↗
PubMed1992

Helical structure of P pili from Escherichia coli. Evidence from X-ray fiber diffraction and scanning transmission electron microscopy.

The structure of the P pili from Escherichia coli has been studied using X-ray fiber diffraction and scanning transmission electron microscopy (STEM). Analysis of the fiber diffraction data indicates that the pili are constituted largely of structural subunits arranged helically with approximately 33 subunits in 10 turns in an axial repeat of 244.5 +/- 1.8 A. Radial electron density distributions calculated from equatorial diffraction data and STEM data indicate that the pili are about 65 A in diameter with a small central cavity roughly 15 A across. The principal protein component of the pili is PapA, which has a molecular weight of 16.5 kDa. Assuming that each subunit consists of a single PapA molecule, the mass-per-unit-length of the pili predicted from the X-ray data is 2.23 kDa/A. Measurements of mass-per-unit-length were also made through the analysis of STEM images. These measurements indicate a value of 2.13 +/- 0.14 kDa/A. STEM images demonstrated the presence of thin, thread-like structures emerging from the ends of pili and spanning breaks in the pili structure. These structures, which have been observed under other conditions, have been termed fibrillae. In the STEM images the fibrillae appear about 20 A in diameter. The mass-per-unit-length of the fibrillae was estimated using the STEM data to be 0.4 kDa/A. These data are consistent with the fibrillae representing an unwound or unraveled form of the pili proteins overstretched to about five times the length they would have in the intact pili.

Bacterial Proteins↗
PubMed1993

Bacteriophage PRD1 proteins: cross-linking and scanning transmission electron microscopy analysis.

Bacteriophage PRD1, a double-stranded DNA virus infecting Escherichia coli, has a membrane inside the protein capsid. Chemical cross-linking and scanning transmission electron microscopy showed that the multimeric major coat protein (P3) exists in a trimeric form. Cross-linking revealed, in addition, that protein P11, located between the protein coat and the membrane, exists also as a homotrimer. Minor protein P7 was associated with the major coat protein P3. Under nonreducing conditions the infectivity proteins P16 and P18 formed homomultimeric complexes which were dissociated upon addition of 2-mercaptoethanol.

Capsid↗
PubMed1984

Methylenetetrahydrofolate reductase. Evidence for spatially distinct subunit domains obtained by scanning transmission electron microscopy and limited proteolysis.

Scanning transmission electron microscopy of individual unfixed molecules of methylenetetrahydrofolate reductase has been used to determine the molecular mass distribution of the protein. Methylenetetrahydrofolate reductase, which has a subunit molecular mass of 77 kilodaltons, was found to exist predominantly as a dimer with an apparent molecular mass of 136 +/- 29 kilodaltons. The mass distribution of the enzyme molecules was unchanged in the presence of the allosteric inhibitor S-adenosylmethionine. Examination of negatively stained protein molecules suggested that each subunit of the dimer consists of two globular domains of approximately equal size. Limited proteolysis of the enzyme by trypsin gave results which were entirely consistent with the presence of two domains per subunit. In the presence of 1% trypsin, the enzyme was cleaved into two fragments. The masses of these fragments were 39 and 36 kilodaltons as assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Tryptic cleavage did not lead to loss of NADPH-menadione or NADPH-methylenetetrahydrofolate oxidoreductase activity, and the flavin prosthetic group remained bound to the protein. However, the cleaved protein was completely desensitized with respect to inhibition by S-adenosylmethionine. These results suggest that each subunit of methylenetetrahydrofolate reductase contains two domains and that allosteric inhibition requires specific interactions between these domains. The region between these two domains appears to be very sensitive to proteolysis, while the domains themselves are relatively resistant to further degradation.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1985

Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.

Capsid↗
PubMed1987

Chromatin higher-order structure studied by neutron scattering and scanning transmission electron microscopy.

Neutron scattering in solution and scanning transmission electron microscopy were simultaneously done on chicken erythrocyte chromatin at various salt and magnesium concentrations. We show that chromatin is organized into a higher-order structure even at low ionic strength and that the mass per unit length increases continuously as a function of salt concentration, reaching a limiting value of between six and seven nucleosomes per 11 nm. There is no evidence of a transition from a 10-nm to a 30-nm fiber. Fiber diameter is correlated with mass per unit length, showing that both increase during condensation. We also find that there is no essential difference between the mass per unit length measured by scanning transmission electron microscopy and neutron scattering in solution, showing that the ordered regions seen in micrographs are representative of chromatin in solution.

Animals↗
PubMed1991

Morphologic changes in the human amnion epithelium that accompany labor as seen with scanning and transmission electron microscopy.

Scanning and transmission electron microscopy were used to assess the influence of normal, active labor on the ultrastructure of the human amnion epithelial membrane. Amnion membranes (reflected and placental portions) were obtained from patients either in active labor who were delivered vaginally or by cesarean section after 6 to 12 hours of labor or from patients who underwent elective cesarean section before clinical signs of overt labor. Scanning electron microscopy revealed that reflected amnion membranes that were obtained from patients who were not in labor consisted of a uniform single layer of epithelial cells with numerous microvilli on the apical surface and closely associated cellular borders. In contrast, amnion membranes that were obtained from patients who were in labor consisted of a single layer of epithelial cells, which was interrupted by wide intercellular gaps and extracellular extrusions. Transmission electron microscopy showed that intercellular junctions tended to be less complex in patients who were in labor versus patients who were not in labor. Although lipid droplets were prevalent in both patient groups, specimens that were obtained from patients who were in labor had more lipid droplets per cell than specimens from patients who were not in labor. These results support the theory that the complex biochemical events that culminate in parturition are accompanied and/or preceded by demonstrable morphologic changes in the amnion membrane.

Amnion↗
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↗
PubMed1985

Quaternary structure of pyruvate dehydrogenase complex from Escherichia coli.

The pyruvate dehydrogenase complex of Escherichia coli and subcomplexes derived from it by selective removal of component enzymes have been subjected to quaternary structural analysis by scanning transmission electron microscopy. Scanning transmission electron microscopic images of the intact complex (E1E2E3), the dihydrolipoyl transacetylase-dihydrolipoyl dehydrogenase (E2E3) subcomplex, and the E2 core enzyme appear as cubic particles in various orientations. Mass distributions within this complex and its subcomplexes have been determined by radial mass analysis of similarly oriented scanning transmission electron microscopic images of each type. The data show that mass attributable to dihydrolipoyl dehydrogenase (E3) is well integrated into the structural framework of the E2 core, dihydrolipoyl transacetylase, whereas mass attributable to pyruvate dehydrogenase (E1) is located about the periphery of the core enzyme. The mass distributions are fully consistent with a structural model in which 6 E3 dimers are integrated into the six faces of the cubic E2 core, and 12 E1 dimers are associated along the 12 edges of the core enzyme.

Escherichia coli↗