Small colloidal gold conjugated to fab fragments or to immunoglobulin g as high-resolution labels for electron microscopy: A technical overview

1990 ◽  
Vol 14 (4) ◽  
pp. 313-323 ◽  
Author(s):  
W. Baschong ◽  
N. G. Wrigley
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Immunoglobulin G ◽  
Colloidal Gold ◽  
Fab Fragments

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

scholarly journals A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.

1992 ◽  
Vol 40 (2) ◽  
pp. 177-184 ◽  
Author(s):  
J F Hainfeld ◽  
F R Furuya
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Light Microscopy ◽  
Colloidal Gold ◽  
Gold Cluster ◽  
Covalent Attachment ◽  
Gold Compound ◽  
Fab Fragments ◽  
Gold Core ◽  
Antigenic Sites

A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of 7.0 nm. The cluster's visibility can be enhanced with silver development for use in EM or light microscopy for histological purposes, or to detect less than or equal to 0.2 pg of antigen in immunoblots. By using a gold compound with covalent attachment, a number of advantages over colloidal gold probes are realized, including better resolution, stability, uniformity, sensitivity, and complete absence of aggregation; its small size should also improve penetration and more quantitative labeling of antigenic sites.

A Preparation of High Resolution Standards for Electron Microscopy. Part II

1971 ◽  
Vol 29 ◽  
pp. 160-161
Author(s):  
Akira Tanaka ◽  
David F. Harling
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Carbon Black ◽  
Single Crystal ◽  
Dark Field ◽  
Graphitized Carbon Black ◽  
Graphitized Carbon ◽  
Dark Field Imaging ◽  
Crystal Films ◽  
Micro Holes

In the previous paper, the author reported on a technique for preparing vapor-deposited single crystal films as high resolution standards for electron microscopy. The present paper is intended to describe the preparation of several high resolution standards for dark field microscopy and also to mention some results obtained from these studies. Three preparations were used initially: 1.) Graphitized carbon black, 2.) Epitaxially grown particles of different metals prepared by vapor deposition, and 3.) Particles grown epitaxially on the edge of micro-holes formed in a gold single crystal film.The authors successfully obtained dark field micrographs demonstrating the 3.4Å lattice spacing of graphitized carbon black and the Au single crystal (111) lattice of 2.35Å. The latter spacing is especially suitable for dark field imaging because of its preparation, as in 3.), above. After the deposited film of Au (001) orientation is prepared at 400°C the substrate temperature is raised, resulting in the formation of many square micro-holes caused by partial evaporation of the Au film.

Structural analysis of the surface-layer protein of spirillum serpens by high-resolution electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 738-739
Author(s):  
W. H. Wu ◽  
R. M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
Surface Layer ◽  
Structural Analysis ◽  
High Resolution ◽  
Low Dose ◽  
High Resolution Electron Microscopy ◽  
Optical Diffraction ◽  
Surface Layer Protein ◽  
Morphological Unit ◽  
Resolution Electron

Spirillum serpens possesses a surface layer protein which exhibits a regular hexagonal packing of the morphological subunits. A morphological model of the structure of the protein has been proposed at a resolution of about 25 Å, in which the morphological unit might be described as having the appearance of a flared-out, hollow cylinder with six ÅspokesÅ at the flared end. In order to understand the detailed association of the macromolecules, it is necessary to do a high resolution structural analysis. Large, single layered arrays of the surface layer protein have been obtained for this purpose by means of extensive heating in high CaCl2, a procedure derived from that of Buckmire and Murray. Low dose, low temperature electron microscopy has been applied to the large arrays.As a first step, the samples were negatively stained with neutralized phosphotungstic acid, and the specimens were imaged at 40,000 magnification by use of a high resolution cold stage on a JE0L 100B. Low dose images were recorded with exposures of 7-9 electrons/Å2. The micrographs obtained (Fig. 1) were examined by use of optical diffraction (Fig. 2) to tell what areas were especially well ordered.

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

High resolution surface structure of foot-and-mouth disease virus

1978 ◽  
Vol 36 (2) ◽  
pp. 376-377
Author(s):  
S. S. Breese ◽  
H. L. Bachrach
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Surface Structure ◽  
Disease Virus ◽  
Potassium Phosphate ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Physical Measurements ◽  
Mouth Disease Virus ◽  
Foot And Mouth

Models for the structure of foot-and-mouth disease virus (FMDV) have been proposed from chemical and physical measurements (Brown, et al., 1970; Talbot and Brown, 1972; Strohmaier and Adam, 1976) and from rotational image-enhancement electron microscopy (Breese, et al., 1965). In this report we examine the surface structure of FMDV particles by high resolution electron microscopy and compare it with that of particles in which the outermost capsid protein VP3 (ca. 30, 000 daltons) has been split into smaller segments, two of which VP3a and VP3b have molecular weights of about 15, 000 daltons (Bachrach, et al., 1975).Highly purified and concentrated type A12, strain 119 FMDV (5 mg/ml) was prepared as previously described (Bachrach, et al., 1964) and stored at 4°C in 0. 2 M KC1-0. 5 M potassium phosphate buffer at pH 7. 5. For electron microscopy, 1. 0 ml samples of purified virus and trypsin-treated virus were dialyzed at 4°C against 0. 2 M NH4OAC at pH 7. 3, deposited onto carbonized formvar-coated copper screens and stained with phosphotungstic acid, pH 7. 3.

High Resolution Study of Polytypism: Application to SiC and Au3 Mn

1982 ◽  
Vol 40 ◽  
pp. 540-541
Author(s):  
G. Van Tendeloo ◽  
J. Van Landuyt ◽  
S. Amelinckx
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Stacking Sequence ◽  
X Ray ◽  
Powerful Technique ◽  
Resolution Study ◽  
The Ideal

Polytypism has been studied for a number of years and a wide variety of stacking sequences has been detected and analysed. SiC is the prototype material in this respect; see e.g. Electron microscopy under high resolution conditions when combined with x-ray measurements is a very powerful technique to elucidate the correct stacking sequence or to study polytype transformations and deviations from the ideal stacking sequence.

Stereo Electron Microscopy Applied to High Resolution Freeze-Etch Replicas

1970 ◽  
Vol 28 ◽  
pp. 306-307
Author(s):  
L. Andrew Staehelin
Keyword(s):  
Electron Microscopy ◽  
Plastic Deformation ◽  
High Resolution ◽  
Smooth Surfaces ◽  
Small Particles ◽  
Membrane Surfaces ◽  
Wide Acceptance ◽  
New Information ◽  
Number Of Particles ◽  
Small Holes

Freeze-etched membranes usually appear as relatively smooth surfaces covered with numerous small particles and a few small holes (Fig. 1). In 1966 Branton (1“) suggested that these surfaces represent split inner mem¬brane faces and not true external membrane surfaces. His theory has now gained wide acceptance partly due to new information obtained from double replicas of freeze-cleaved specimens (2,3) and from freeze-etch experi¬ments with surface labeled membranes (4). While theses studies have fur¬ther substantiated the basic idea of membrane splitting and have shown clearly which membrane faces are complementary to each other, they have left the question open, why the replicated membrane faces usually exhibit con¬siderably fewer holes than particles. According to Branton's theory the number of holes should on the average equal the number of particles. The absence of these holes can be explained in either of two ways: a) it is possible that no holes are formed during the cleaving process e.g. due to plastic deformation (5); b) holes may arise during the cleaving process but remain undetected because of inadequate replication and microscope techniques.

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