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.

Electron Microscopic Observation of the Longitudinal Organization of Poly (p-Phenylene Terephthalamide) Fibers

1982 ◽  
Vol 40 ◽  
pp. 680-681
Author(s):  
Li Li-Sheng ◽  
L.F. Allard ◽  
W.C. Bigelow
Keyword(s):  
Electron Microscopy ◽  
Mechanical Properties ◽  
Scanning Electron Microscopy ◽  
Microscopic Observation ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
Aromatic Polyamides ◽  
Radial Arrangement ◽  
Scanning Electron ◽  
Better Than

The aromatic polyamides form a class of fibers having mechanical properties which are much better than those of aliphatic polyamides. Currently, the accepted morphology of these fibers as proposed by M.G. Dobb, et al. is a radial arrangement of pleated sheets, with the plane of the pleats parallel to the axis of the fiber. We have recently obtained evidence which supports a different morphology of this type of fiber, using ultramicrotomy and ion-thinning techniques to prepare specimens for transmission and scanning electron microscopy.

Reconstruction of Two-Dimensional Projections of GP 32*I

1981 ◽  
Vol 39 ◽  
pp. 38-39
Author(s):  
H.A. Cohen ◽  
W. Chiu ◽  
J. Hosoda
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Uranyl Acetate ◽  
Distilled Water ◽  
Acetate Solution ◽  
Two Dimensional ◽  
Parent Molecule ◽  
T4 Bacteriophage ◽  
Electron Imaging ◽  
Better Than

GP 32 (molecular weight 35000) is a T4 bacteriophage protein that destabilizes the DNA helix. The fragment GP32*I (77% of the total weight), which destabilizes helices better than does the parent molecule, crystallizes as platelets thin enough for electron diffraction and electron imaging. In this paper we discuss the structure of this protein as revealed in images reconstructed from stained and unstained crystals.Crystals were prepared as previously described. Crystals for electron microscopy were pelleted from the buffer suspension, washed in distilled water, and resuspended in 1% glucose. Two lambda droplets were placed on grids over freshly evaporated carbon, allowed to sit for five minutes, and then were drained. Stained crystals were prepared the same way, except that prior to draining the droplet, two lambda of aqueous 1% uranyl acetate solution were applied for 20 seconds. Micrographs were produced using less than 2 e/Å2 for unstained crystals or less than 8 e/Å2 for stained crystals.

Structural Studies on the Eukaryotic Ribosome

1990 ◽  
Vol 48 (1) ◽  
pp. 256-257
Author(s):  
Adriana Verschoor ◽  
Ronald Milligan ◽  
Suman Srivastava ◽  
Joachim Frank
Keyword(s):  
Chick Embryo ◽  
3D Reconstruction ◽  
Single Particle ◽  
Mass Density ◽  
Particle Surface ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
Eukaryotic Ribosome ◽  
Negative Stain ◽  
Reconstruction Methods

We have studied the eukaryotic ribosome from two vertebrate species (rabbit reticulocyte and chick embryo ribosomes) in several different electron microscopic preparations (Fig. 1a-d), and we have applied image processing methods to two of the types of images. Reticulocyte ribosomes were examined in both negative stain (0.5% uranyl acetate, in a double-carbon preparation) and frozen hydrated preparation as single-particle specimens. In addition, chick embryo ribosomes in tetrameric and crystalline assemblies in frozen hydrated preparation have been examined. 2D averaging, multivariate statistical analysis, and classification methods have been applied to the negatively stained single-particle micrographs and the frozen hydrated tetramer micrographs to obtain statistically well defined projection images of the ribosome (Fig. 2a,c). 3D reconstruction methods, the random conical reconstruction scheme and weighted back projection, were applied to the negative-stain data, and several closely related reconstructions were obtained. The principal 3D reconstruction (Fig. 2b), which has a resolution of 3.7 nm according to the differential phase residual criterion, can be compared to the images of individual ribosomes in a 2D tetramer average (Fig. 2c) at a similar resolution, and a good agreement of the general morphology and of many of the characteristic features is seen.Both data sets show the ribosome in roughly the same ’view’ or orientation, with respect to the adsorptive surface in the electron microscopic preparation, as judged by the agreement in both the projected form and the distribution of characteristic density features. The negative-stain reconstruction reveals details of the ribosome morphology; the 2D frozen-hydrated average provides projection information on the native mass-density distribution within the structure. The 40S subunit appears to have an elongate core of higher density, while the 60S subunit shows a more complex pattern of dense features, comprising a rather globular core, locally extending close to the particle surface.

The NCEM One-Angstrom Microscope Project Reaches 0.89Å Resolution

2000 ◽  
Vol 6 (S2) ◽  
pp. 1192-1193 ◽  
Author(s):  
Michael A. O'Keefe
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Image Reconstruction ◽  
Structural Information ◽  
Computer Software ◽  
Image Simulation ◽  
Resolution Limit ◽  
Transmission Electron ◽  
Better Than

Transmission electron microscopy to a resolution of 0.89Å has been achieved at the National Center for Electron Microscopy and is available to electron microscopists who have a requirement for this level of resolution. Development of this capability commenced in 1993, when the National Center for Electron Microscopy agreed to fund a proposal for a unique facility, a one- Ångstrom microscope (OÅM).2 The OÅM project provides materials scientists with transmission electron microscopy at a resolution better than one Angstrom by exploiting the significantly higher information limit of a FEG-TEM over its Scherzer resolution limit. To turn the misphased information beyond the Scherzer limit into useful resolution, the OÅM requires extensive image reconstruction. One method chosen was reconstruction from off-axis holograms; another was reconstruction from focal series of underfocused images. The OÅM is then properly a combination of a FEG-TEM (a CM300FEG-UT) together with computer software able to generate sub-Ångstrom images from experimental images obtained on the FEG-TEM.Before the advent of the OÅM, NCEM microscopists relied on image simulation to obtain structural information beyond the TEM resolution limit.

Problems in Myogenesis Terminology in Electron Microscopy

1967 ◽  
Vol 25 ◽  
pp. 38-39
Author(s):  
P.E. Conen ◽  
J.U. Balis ◽  
C.D. Bell
Keyword(s):  
Electron Microscopy ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
Accurate Identification ◽  
Microscopic Studies ◽  
A Cell ◽  
Lead Hydroxide ◽  
Developing Muscle

Myogenesis in man was studied using muscle from 19 fetuses of 8 to 16 weeks gestation which were processed with standard osmium-Epon or glutaraldehyde-osmium-Epon schedules and sections were stained in uranyl acetate and/or lead hydroxide. Particular emphasis was given during this study to presence of basement membrane and myofilaments as additional aids in classification of cell types present in developing muscle.Electron microscopy permits accurate identification of fibroblasts and early cells of muscle series and has been used in studies of myogenesis in chick, and rat. Light microscopy definitions for premyoblasts and myoblasts, and for myocytes at the myotube and muscle fiber stages of development are difficult to apply to electron microscopic studies without modification. For example the term myoblast was used differently by Tello, Katznelson and Boyd to designate a cell destined to become muscle but not recognizable as a muscle cell.

A Modified Technic of Staining Elastic Tissue for Electron Microscopic Study

1975 ◽  
Vol 33 ◽  
pp. 626-627
Author(s):  
J.A. Nordquist ◽  
K. Chrysant ◽  
A.K. Mandal
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Renal Tissue ◽  
Uranyl Acetate ◽  
Elastic Tissue ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Modified Method ◽  
Tetraphenyl Porphyrin ◽  
Normal Rat

By electron microscopy elastic tissue appear electrolucent in osmium fixed unstained grids as well as grids stained with uranyl acetate and lead citrate (UA + LC). Albert and Fleischer have studied aorta of mice with metalloporphyrins imparting conspicuous electron density to the elastic tissue. We are reporting here a modified method of electron microscopic (EM) study of the elastic tissue using metalloporhyrin, silver tetraphenyl porphyrin sulfonate (STPPS).We have studied the renal arterioles of rats and human in normal and diseased states. Elastic tissue of the aorta from young normal rat served as control for this study. Renal and aortic tissues were fixed in 4 percent glutaraldehyde, post fixed in 1 percent osmium tetroxide and embedded in spurr (blocks). From the blocks of renal tissue, 0.5 μ sections were cut, stained with methylene blue and azure II and studied by light microscopy.

scholarly journals Retrospective demonstration of endogenous peroxidase activity in plastic-embedded tissues conventionally prepared for electron microscopy.

1981 ◽  
Vol 29 (7) ◽  
pp. 874-876 ◽  
Author(s):  
M Del Cerro ◽  
J P Cogen ◽  
C Del Cerro
Keyword(s):  
Electron Microscopy ◽  
Peroxidase Activity ◽  
Pigment Epithelium ◽  
Sodium Ethoxide ◽  
Retinal Pigment ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Microscopical Examination ◽  
Electron Microscopic ◽  
Endogenous Peroxidase

A procedure is described that permits retrospective demonstration of intracellular endogenous peroxidase activity in tissue conventionally prepared for electron microscopy, i.e., doubly fixed with aldehydes and osmium tetroxide, "stained" in block with uranyl acetate, and embedded in epoxy resins. Using sodium ethoxide, plastic was removed from 1 micrometer sections; subsequently, the sections were incubated for 20 min in diaminobenzidine solution (44 mg/100 ml) made in acetate-citric acid buffer, pH 5.6, with 0.01% hydrogen peroxide. After this treatment, the sections were rinsed, dehydrated, and mounted. Cell types known to have endogenous peroxidase activity (red blood cells, macrophages, and retinal pigment epithelium cells in our preparations) show positive granules in their cytoplasm--control sections were uniformly negative. This method, which could also be used prospectively, cytochemically demonstrates endogenous peroxidase activity upon optical microscopical examination of the treated tissues; correlative electron microscopic studies may be performed on the same tissue block, or even adjacent sections.

The Connective Tissue Matrix of Cartilage as Revealed by Standard Fixation, Ruthenium Staining, High-Pressure Freezing, and Embedding in Water Soluble Media: A Comparison of Methods

1990 ◽  
Vol 48 (2) ◽  
pp. 326-327
Author(s):  
Douglas R. Keene
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Ruthenium Red ◽  
Water Soluble ◽  
High Pressure Freezing ◽  
Electron Microscopic ◽  
Microscopic Evaluation ◽  
Embedding Medium

Proteoglycan is a major component of the cartilage extracellular matrix, and the overall structure of this anionic molecule is highly dependent on the hydrated environment of cartilage. Without specific stabilization, proteoglycans are extracted or collapsed during deydration while processing for electron microscopy. The purpose of these experiments is to determine a method by which the structure of proteoglycans might be stabilized for electron microscopic evaluation.Chick sternal cartilage was prepared for transmission electron microscopy by the following methods and the resultant tissue ultrastructure compared: A) 1.5/1.5% gluteraldehyde/paraformaldehyde and 1% OsO4 fixation, dehydration in ethanol, propylene oxide, and embedding in Spurrs epoxy B) Fixation as in (A) directly followed by infiltration and embedding in Hexamethylol-melamine-methyl-ether (a water soluble embedding medium) trade name “nanoplast” C) Fixation by high pressure freezing followed by freeze substitution in acetone/OsO4 prior to embedding in epon 812. In variations of methods A and B above, ruthenium red (RR, 1500 ppm) or ruthenium hexamine trichloride (RHT, 6000 ppm) were added to the primary and secondary fixatives. All tissue sections were stained in uranyl acetate and lead citrate.

Organs of a Baikal seal affected by a morbillivirus

1990 ◽  
Vol 48 (3) ◽  
pp. 968-969
Author(s):  
O.I. Belykh ◽  
Ye.V. Likhoshway ◽  
Yu.V. Solodun ◽  
O.A. Goldberg ◽  
V.P. Kumarev
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Present Report ◽  
Protein A ◽  
Uranyl Acetate ◽  
Kidney Cells ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Liver And Kidney ◽  
Ultrathin Sections

The population of Baikal seals Phoca sibirica has been plagued in 1987-88 by an unknown disease. Oligonucleotide probing of nucleic acids isolated from tissues of ill and dead animals, as well as immunological evidence and clinical data suggested that seals were infected by a morbillivirus. Morbillivirus antigen has been vizualized in dead seal tissues by immunoelectron microscopy (preembedding technique).The present report gives outline of electron microscopic studies of the tissues of infected Baikal seals. Morbillivirus antigens were vizualized as clusters of gold spheres by postembedding technique with monoclonal antibodies against measles virus and protein A-colloid gold conjugates in nuclei and cytoplasm of liver and kidney cells. Some clusters were associated with virus-like particles having a diameter of 80-100 nm. Electron microscopy of ultrathin sections stained with uranyl acetate revealed nucleocapsides having length of up to 1400 nm, and a diameter of 13-17 nm, morphologically similar to measles and seals distemper virus.

scholarly journals EVIDENCE FOR CHANGES IN PROTEIN POLYSACCHARIDE ASSOCIATED WITH THE ONSET OF CALCIFICATION IN CARTILAGE

1968 ◽  
Vol 39 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Victor J. Matukas ◽  
George A. Krikos
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Marked Decrease ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Calcified Cartilage ◽  
The Matrix ◽  
Microscopic Studies ◽  
Matrix Components

Past work has suggested that protein polysaccharide may play a role in the calcification of cartilage. Recent electron microscopic studies on noncalcified cartilage have indicated that protein polysaccharide in cartilage matrix is represented by granules associated with collagen fibers. The present work has been designed for comparison of the matrix of noncalcified cartilage to that of calcified cartilage, with particular reference to these granules. Small blocks of tibia from 16-day embryos were fixed in cacodylate-buffered glutaraldehyde and postfixed in either phosphate- or Veronal-buffered osmium tetroxide. Special care was taken to maintain the pH above 7.0 at all times. For electron microscopy the tissues were dehydrated, embedded in Epon 812, sectioned, and stained with uranyl acetate or lead citrate. A marked decrease in the size of granules in the matrix of calcified cartilage compared to noncalcified cartilage was noted. Associated with the decrease in the size of granules was a condensation of matrix components and the presence of an amorphous electron-opaque material that was not seen in noncalcified areas. These results are interpreted to represent either a drop in concentration or a change in state of protein polysaccharide with the onset of calcification in cartilage.

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