Thin Mica for Electron Microscopy

1964 ◽  
Vol 35 (1) ◽  
pp. 124-124 ◽  
Author(s):  
Ethel L. Fontanella
Keyword(s):  
Electron Microscopy ◽  
Thin Mica

Three Methods For the Preparation of High-Resolution Freeze-Dried Specimens in a Freeze-Etch Module

1973 ◽  
Vol 31 ◽  
pp. 338-339
Author(s):  
Russell L. Steere
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Freeze Drying ◽  
High Vacuum ◽  
Distilled Water ◽  
Specimen Temperature ◽  
Particulate Suspension ◽  
Freeze Dried ◽  
Thin Mica

The first satisfactory freeze-dry procedure for electron microscopy was described by Williams. An improvement permitted the shadowing of specimens in the same system where freeze-drying was accomplished. Our freeze- etch module is ideal also for the preparation of freeze-dry specimens and high-resolution specimens have been prepared by the 3 different procedures.Procedure #1: A particulate suspension in distilled water or a volatile buffer is sprayed onto a thin mica sheet held down with high vacuum grease to a flat-topped specimen cap clamped onto the specimen stage (all pre-cooled to −150°C). Freeze-drying is accomplished with a vacuum of 2 × 10−6 Torr and specimen temperature of −98 to −75° (approximately 15 min).

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

Immune Electron Microscopy (IEM) of a Canine Adeno-Associated Virus

1974 ◽  
Vol 32 ◽  
pp. 256-257
Author(s):  
Gunter F. Thomas ◽  
M. David Hoggan
Keyword(s):  
Electron Microscopy ◽  
Cell Cultures ◽  
Complement Fixation ◽  
Hepatitis Virus ◽  
Wide Distribution ◽  
Immune Electron Microscopy ◽  
Adeno Associated Virus ◽  
Virus Like Particle ◽  
Dog Kidney ◽  
Green Monkeys

In 1968, Sugimura and Yanagawa described a small 25 nm virus like particle in association with the Matsuda strain of infectious canine hepatitis virus (ICHV). Domoto and Yanagawa showed that this particle was dependent on ICHV for its replication in primary dog kidney cell cultures (PDK) and was resistant to heating at 70°C for 10 min, and concluded that it was a canine adeno-associated virus (CAAV). Later studies by Onuma and Yanagawa compared CAAV with the known human serotypes (AAV 1, 2, 3) and AAV-4, known to be associated with African Green Monkeys. Using the complement fixation (CF) test, they found that CAAV was serologically related to AAV-3 and had wide distribution in the dog population of Japan.

High Voltage Electron Microscopy of Ion-Milled Dental Enamel

1974 ◽  
Vol 32 ◽  
pp. 60-61
Author(s):  
L. D. Ackerman ◽  
S. H. Y. Wei
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Third Molar ◽  
Polarized Light ◽  
Dental Enamel ◽  
Longitudinal Section ◽  
Ion Milling ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron

Mature human dental enamel has presented investigators with several difficulties in ultramicrotomy of specimens for electron microscopy due to its high degree of mineralization. This study explores the possibility of combining ion-milling and high voltage electron microscopy as a means of circumventing the problems of ultramicrotomy.A longitudinal section of an extracted human third molar was ground to a thickness of about 30 um and polarized light micrographs were taken. The specimen was attached to a single hole grid and thinned by argon-ion bombardment at 15° incidence while rotating at 15 rpm. The beam current in each of two guns was 50 μA with an accelerating voltage of 4 kV. A 20 nm carbon coating was evaporated onto the specimen to prevent an electron charge from building up during electron microscopy.

Pathogenesis of Human Hair Defects

1974 ◽  
Vol 32 ◽  
pp. 12-13
Author(s):  
P.S. Porter ◽  
T. Aoyagi ◽  
R. Matta
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Human Hair ◽  
Standard Techniques ◽  
Scanning Electron

Using standard techniques of scanning electron microscopy (SEM), over 1000 human hair defects have been studied. In several of the defects, the pathogenesis of the abnormality has been clarified using these techniques. It is the purpose of this paper to present several distinct morphologic abnormalities of hair and to discuss their pathogenesis as elucidated through techniques of scanning electron microscopy.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

Quaternary structure of Limulus polyphemus hemocyanin

1978 ◽  
Vol 36 (2) ◽  
pp. 176-177
Author(s):  
T. Wichertjes ◽  
E.J. Kwak ◽  
E.F.J. Van Bruggen
Keyword(s):  
Electron Microscopy ◽  
Functional Properties ◽  
Horseshoe Crab ◽  
Temperature Jump ◽  
Quaternary Structure ◽  
Crystallographic Analysis ◽  
Limulus Polyphemus ◽  
Oxygen Binding ◽  
X Ray ◽  
Intact Molecule

Hemocyanin of the horseshoe crab (Limulus polyphemus) has been studied in nany ways. Recently the structure, dissociation and reassembly was studied using electron microscopy of negatively stained specimens as the method of investigation. Crystallization of the protein proved to be possible and X-ray crystallographic analysis was started. Also fluorescence properties of the hemocyanin after dialysis against Tris-glycine buffer + 0.01 M EDTA pH 8.9 (so called “stripped” hemocyanin) and its fractions II and V were studied, as well as functional properties of the fractions by NMR. Finally the temperature-jump method was used for assaying the oxygen binding of the dissociating molecule and of preparations of isolated subunits. Nevertheless very little is known about the structure of the intact molecule. Schutter et al. suggested that the molecule possibly consists of two halves, combined in a staggered way, the halves themselves consisting of four subunits arranged in a square.

Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 166-167 ◽  
Author(s):  
G. Stöffler ◽  
R.W. Bald ◽  
J. Dieckhoff ◽  
H. Eckhard ◽  
R. Lührmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Spatial Arrangement ◽  
Small Subunit ◽  
Antibody Binding ◽  
Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

Immune electron microscopy of haemocyanins from two southern african whelks

1978 ◽  
Vol 36 (2) ◽  
pp. 158-159
Author(s):  
Linda M. Stannard ◽  
Margaret Lennon
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Outer Ring ◽  
Immune Electron Microscopy ◽  
Intertidal Zones ◽  
Central Belt ◽  
Cape Peninsula ◽  
Circular Contour

Burnupena cincta and Fusus verruculatus are two whelks which inhabit the intertidal zones of the Cape Peninsula shore. Their respiratory pigments, or haemocyanins, are morphologically similar in structure (Figs. 1 and 2) and appear in the electron microscope as short cylindrical rods about 34 nm in diameter and 36 nm high. Viewed side-on the molecules show regular banding suggesting a structure composed of six equidistant rings of sub-units. Occasionally the particles have the appearance of possessing a central “belt” in the position of the 3rd and 4th rows of sub-units. End-on views of the haemocyanin molecules show a circular contour with a dense outer ring and a less dense inner ring in which 10 definite sub-units may frequently be distinguished. A number of molecules display an extra central inner component which appears either as a diffuse plug or as a discrete ring-shaped core ± 8 nm in diameter.

Localization of immunolabels by electron microscopy and image averaging

1983 ◽  
Vol 41 ◽  
pp. 282-283
Author(s):  
J. Frank ◽  
P.-Y. Sizaret ◽  
A. Verschoor ◽  
J. Lamy
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Attachment Site ◽  
Uranyl Acetate ◽  
Limulus Polyphemus ◽  
Resolution Limit ◽  
Electron Microscopic ◽  
Image Averaging ◽  
Top View ◽  
Better Than

The accuracy with which the attachment site of immunolabels bound to macromolecules may be localized in electron microscopic images can be considerably improved by using single particle averaging. The example studied in this work showed that the accuracy may be better than the resolution limit imposed by negative staining (∽2nm).The structure used for this demonstration was a halfmolecule of Limulus polyphemus (LP) hemocyanin, consisting of 24 subunits grouped into four hexamers. The top view of this structure was previously studied by image averaging and correspondence analysis. It was found to vary according to the flip or flop position of the molecule, and to the stain imbalance between diagonally opposed hexamers (“rocking effect”). These findings have recently been incorporated into a model of the full 8 × 6 molecule.LP hemocyanin contains eight different polypeptides, and antibodies specific for one, LP II, were used. Uranyl acetate was used as stain. A total of 58 molecule images (29 unlabelled, 29 labelled with antl-LPII Fab) showing the top view were digitized in the microdensitometer with a sampling distance of 50μ corresponding to 6.25nm.

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