scholarly journals Demonstration of antigens at both sides of plasma membranes in one coincident electron microscopic image: a double-immunogold replica study of virus-infected cells.

1988 ◽  
Vol 36 (8) ◽  
pp. 1015-1021 ◽  
Author(s):  
G Rutter ◽  
W Bohn ◽  
H Hohenberg ◽  
K Mannweiler
Keyword(s):  
High Resolution ◽  
Measles Virus ◽  
Plasma Membranes ◽  
Protein A ◽  
Three Dimensional ◽  
Electron Microscopic ◽  
Cytoplasmic Surface ◽  
Critical Point Drying ◽  
Electron Microscopic Image ◽  
Infected Cells

We present here a procedure for obtaining high-resolution topographical information about the spatial distribution of antigens at both sides of isolated plasma membranes. HeLa cells grown on coverslips and infected with measles virus served as a model system. Virus glycoproteins appearing at the cell surface were demonstrated by tagging them with rabbit anti-measles antibodies and protein A-gold probes. Cells were stabilized with tannic acid, covered with a cationized coverslip, and then split in potassium-containing buffer. Membranes adherent to the cationized coverslip were fixed in formaldehyde-glutaraldehyde and reacted with mouse monoclonal antibodies against various structural proteins of measles virus. Antibody binding sites at the cytoplasmic surface were visualized either by the antibody bridge method, using normal mouse Ig coupled to gold colloid of different sizes, or by the peroxidase-antiperoxidase procedure. After osmication and critical point-drying, the cytoplasmic surfaces were replicated by platinum-carbon evaporation and examined by TEM without prior cleaning from biological material. This new method permits concomitant localization of antigens present at the inner and outer leaflets of the plasma membrane, and provides high-resolution information about the three-dimensional organization of the cytoplasmic surface.

scholarly journals High-resolution three-dimensional views of membrane-associated clathrin and cytoskeleton in critical-point-dried macrophages.

1983 ◽  
Vol 97 (5) ◽  
pp. 1452-1458 ◽  
Author(s):  
J Aggeler ◽  
R Takemura ◽  
Z Werb
Keyword(s):  
Plasma Membrane ◽  
High Resolution ◽  
Critical Point ◽  
Plasma Membranes ◽  
Cytoplasmic Membrane ◽  
Three Dimensional ◽  
Ventral Surface ◽  
Experimental Conditions ◽  
Membrane Surfaces ◽  
Critical Point Drying

We obtained high-resolution topographical information about the distribution of clathrin and cytoskeletal filaments on cytoplasmic membrane surfaces of macrophages spreading onto glass coverslips by both critical-point drying of broken-open cells and preparation of rotary platinum replicas. Irregular patches of the adherent ventral surface of the plasma membrane were exposed in these cells, and large areas of these exposed membranes were covered with clathrin-coated patches, pits, and vesicles. Various amounts of cytoskeleton were attached to the plasma membranes of these spreading cells, either as distinct starlike foci, or as individual filaments and bundles radiating out from the cytoskeletal meshwork. In newly adherent cells a well developed Golgi-GERL complex, characterized by smooth, dish-like cisternae associated with rough endoplasmic reticulum, was observed. There were many coated vesicles budding off from the Golgi cisternae, and these were predominantly of the large type (150 nm) usually associated with the plasma membrane. In critical-point-dried samples, both cytoskeleton and membranes were preserved in detail comparable to that of quick-frozen samples, after appropriate fixation. Rotary replication of critical-point-dried cells provides a rapid, easily controlled, and generally easy to perform method for obtaining samples of exposed membrane large enough to permit quantification of membrane-associated clathrin and cytoskeleton under various experimental conditions.

High-resolution transmission electron microscopic investigations of molybdenum thin films on faceted α-Al2O3

2005 ◽  
Vol 38 (2) ◽  
pp. 260-265 ◽  
Author(s):  
Leonore Wiehl ◽  
Jens Oster ◽  
Michael Huth
Keyword(s):  
High Resolution ◽  
Three Dimensional ◽  
Epitaxial Relationship ◽  
Electron Microscopic ◽  
Force Microscopy ◽  
Atomic Force ◽  
Transmission Electron ◽  
High Resolution Images ◽  
Α Al2o3 ◽  
Relationship Of

Epitaxially grown Mo films on a faceted corundum (α-Al2O3)mplane were investigated by transmission electron microscopy. Low- and high-resolution images were taken from a cross-section specimen cut perpendicular to the facets. It was possible to identify unambiguously the crystallographic orientation of these facets and explain the considerable deviation (∼10°) of the experimental interfacet angle, as measured with atomic force microscopy (AFM), from the expected value. For the first time, proof is given for a smooth \{10\bar{1}1\} facet and a curvy facet with orientation near to \{10\bar{1}\bar{2}\}. Moreover, the three-dimensional epitaxial relationship of an Mo film on a faceted corundummsurface was determined.

Effect of acoustic overstimulation on the glycocalyx and the ciliary interconnections in the organ of Corti: High resolution scanning electron microscopic investigation

1989 ◽  
Vol 103 (12) ◽  
pp. 1125-1129 ◽  
Author(s):  
M. Takumida ◽  
L. Fredelius ◽  
D. Bagger-Sjöbäck ◽  
Y. Harada ◽  
J. Wersäll
Keyword(s):  
High Resolution ◽  
Three Dimensional ◽  
Organ Of Corti ◽  
Staining Technique ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Outer Hair Cells ◽  
Electron Microscopic ◽  
Acoustic Overstimulation ◽  
Scanning Electron

AbstractChanges in ciliary interconnections in the organ of Corti are described after acoustic overstimulation using a special high resolution scanning electron microscope and tannic acid-osmium staining technique, giving an almost three dimensional view. Guinea pigs were exposed to a 3.85 kHz pure tone at an intensity of 120 dB for 22.5 minutes. The first detectable change was a disarrangement of the cilia with a loosening of the interconnections. The ciliary plasma membrane presented with an abnormally smooth appearance. The tip links connecting the tips of the stereocilia to their taller neighbours were also affected showing elongation or even disappearance. The fine granules which cover the tips of the tallest stereocilia of the outer hair cells were decreased. These findings suggest that acoustic overstimulation may affect the carbohydrate metabolism exceding to degeneration of ciliary interconnections resulting in a disarrangement and detachment of cilia. The tip links, which may participate in sensory cell transduction, seem also to be affected by acoustic overstimulation.

scholarly journals Antisera to gamma-aminobutyric acid. III. Demonstration of GABA in Golgi-impregnated neurons and in conventional electron microscopic sections of cat striate cortex.

1985 ◽  
Vol 33 (3) ◽  
pp. 249-257 ◽  
Author(s):  
P Somogyi ◽  
A J Hodgson
Keyword(s):  
Striate Cortex ◽  
Protein A ◽  
Three Dimensional ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Electron Microscopic ◽  
Golgi Impregnation ◽  
Second Procedure ◽  
Unlabeled Antibody ◽  
Layer Vi

Two methods are described for the immunocytochemical demonstration of immunoreactive gamma-aminobutyric acid (GABA) in the visual cortex of the cat, an area that contains several types of GABAergic neurons and requires combined methods for their characterization. The first method is illustrated by a representative example of a Golgi-impregnated and gold-toned interneuron of the "bitufted" type situated in layer VI and having an ascending axon. After recording the three-dimensional features of the cell, semithin (0.5 micron) sections of the perikaryon were cut and GABA was demonstrated in the cell body by the unlabeled antibody enzyme method. While immunocytochemistry was used to determine the probable transmitter of the neuron, Golgi-impregnation of the same cell was used to identify its neuronal type. Since aldehyde-osmium fixation was used, further electron microscopic (EM) analysis of the neuron's synaptic connections was possible. The second procedure demonstrated GABA in EM sections of aldehyde-osmium-fixed cortex using protein A-gold as an immunocytochemical marker. Immunoreactivity was found in certain neurons, dendrites, axons, and boutons forming type II synaptic contacts that from previous studies have been thought to be GABAergic. Thus ultrastructural analysis using optimal conditions can now be supplemented with the identification of the transmitter in the same section.

scholarly journals Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

1994 ◽  
Vol 126 (4) ◽  
pp. 901-910 ◽  
Author(s):  
T J Deerinck ◽  
M E Martone ◽  
V Lev-Ram ◽  
D P Green ◽  
R Y Tsien ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Substantial Improvement ◽  
Electron Microscopic ◽  
Simple Method ◽  
High Voltage Electron Microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

scholarly journals Immunocytochemical study of the partition and distribution of Sindbis virus glycoproteins in freeze-fractured membranes of infected baby hamster kidney cells.

1985 ◽  
Vol 101 (4) ◽  
pp. 1300-1306 ◽  
Author(s):  
M R Torrisi ◽  
S Bonatti
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Plasma Membranes ◽  
Protein A ◽  
Kidney Cells ◽  
Baby Hamster Kidney ◽  
Immunocytochemical Study ◽  
Viral Glycoproteins ◽  
Infected Cells ◽  
Baby Hamster Kidney Cells

Sindbis virus-infected baby hamster kidney cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies or with conventional lectin label (wheat germ agglutinin) were used in conjunction with colloidal gold-conjugated protein A or ovomucoid, respectively. In addition, intact infected cells were analyzed with both labeling procedures. Experiments with Sindbis infected-chick embryo fibroblast cells were carried out as controls. Viral transmembrane glycoproteins appeared present in freeze-fractured inner and outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes; a clear preferential partition with the exoplasmic faces of all intracellular membranes was observed. By contrast, at the plasma membrane level, Sindbis glycoproteins were found to partition preferentially with the protoplasmic face. It seems likely that this protoplasmic partition is related to the binding with the nucleocapsid that takes place during the budding of the virus. At the cell surface, viral glycoproteins always appeared clustered and were predominantly associated with budding figures: moreover, large portions of the plasma membrane were devoid of both glycoproteins and budding viruses.

The Degradation of the Rhinovirion as Studied By High Resolution Stereo Electron Microscopy

1969 ◽  
Vol 27 ◽  
pp. 404-405
Author(s):  
Jerry D. Reeves ◽  
Heather D. Mayor
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Small Rna ◽  
Viral Genome ◽  
Original Material ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Electron Microscopic Image ◽  
Small Virus ◽  
Acid Stable

Picornaviruses, a group of small RNA-containing viruses are further divided into two sub-groups, the enteroviruses which are acid stable, and the rhinoviruses which suffer marked loss of infectivity at acid pH. We have found a ninety per cent decrease in infectious titer after subjection to pH 5.5 as compared to the titer of the original material at pH 7. 0. Considerable morphological degradation of the virion, as monitored by electron microscopy, has also occurred by pH 5. 5. It appears that capsid breakdown proceeds through loss of a vertex capsomere group followed by release of the viral genome in the form of a ribonucleoprotein strand (Fig. 1). However, the conventional flattened electron microscopic image produced by these small virus particles makes further interpretation at a high resolution extremely difficult.

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