Three-dimensionality and fine scale complementarity of freeze-fracture and freeze-etch specimens as revealed by high resolution stereo electron microscopy

Cryobiology ◽  
1971 ◽  
Vol 8 (4) ◽  
pp. 387
Author(s):  
Russell L. Steere
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Freeze Fracture ◽  
Fine Scale

High Resolution Stereography of Complementary Freeze-Fracture Replicas Reveals the Presence of Depressions Opposite Membrane Associated Particles of Split Membranes

1971 ◽  
Vol 29 ◽  
pp. 442-443
Author(s):  
Russell L. Steere
Keyword(s):  
High Resolution ◽  
Cardiac Muscle ◽  
Electron Micrographs ◽  
Chromic Acid ◽  
Freeze Fracture ◽  
Distilled Water ◽  
Fine Scale ◽  
Acid Cleaning ◽  
Cleaning Solution

Complementary replicas have revealed the fact that the two common faces observed in electron micrographs of freeze-fracture and freeze-etch specimens are complementary to each other and are thus the new faces of a split membrane rather than the original inner and outer surfaces (1, 2 and personal observations). The big question raised by published electron micrographs is why do we not see depressions in the complementary face opposite membrane-associated particles? Reports have appeared indicating that some depressions do appear but complementarity on such a fine scale has yet to be shown.Dog cardiac muscle was perfused with glutaraldehyde, washed in distilled water, then transferred to 30% glycerol (material furnished by Dr. Joaquim Sommer, Duke Univ., and VA Hospital, Durham, N.C.). Small strips were freeze-fractured in a Denton Vacuum DFE-2 Freeze-Etch Unit with complementary replica tooling. Replicas were cleaned in chromic acid cleaning solution, then washed in 4 changes of distilled water and mounted on opposite sides of the center wire of a Formvar-coated grid.

scholarly journals The Importance of High-Resolution Scanning Transmission Electron Microscopy For Fine-Scale Dislocation Analysis

2010 ◽  
Vol 16 (S2) ◽  
pp. 1798-1799
Author(s):  
PJ Phillips ◽  
L Kovarik ◽  
RR Unocic ◽  
D Wei ◽  
D Mourer ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Scanning Transmission Electron Microscopy ◽  
Fine Scale ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

A Freeze Fracture Preparation Chamber Attached to the SEM

1977 ◽  
Vol 35 ◽  
pp. 588-589
Author(s):  
J. B. Pawley ◽  
T. L. Hayes
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Chemical Element ◽  
Fracture Process ◽  
Freeze Fracture ◽  
Element Analysis ◽  
Actual Sample ◽  
Membrane Surfaces ◽  
Subsequent Fracture ◽  
Standard Tool

The freeze fracture technique has developed into a standard tool of biological electron microscopy particularly for the study of membrane surfaces. While permitting high resolution (2.5 nm) study of replicated specimens, freeze fracture has always had certain technical limitations: 1) The carbon and platinum replicas are extremely fragile and much care and patience are required to prepare them for EM observation. This is particularly true when large areas must be replicated or when complementary replicas are required. 2) The carbon and platinum replica despite its very “real” appearance is not the actual sample. The sample itself has been completely destroyed in preparing the replica and can neither be subjected to chemical element analysis nor reprocessed for any sort of subsequent study. 3) The investigator can neither monitor the fracture process nor make a subsequent fracture of the same sample.

High Resolution FESEM and TEM Reveal Bacterial Spore Attachment

2007 ◽  
Vol 13 (4) ◽  
pp. 251-266 ◽  
Author(s):  
Barbara J. Panessa-Warren ◽  
George T. Tortora ◽  
John B. Warren
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Field Emission ◽  
Freeze Fracture ◽  
Bacterial Spore ◽  
Attachment Structures ◽  
Spore Attachment ◽  
Electron Microscopes ◽  
The 1960S ◽  
Field Emission Electron Microscopy

Transmission electron microscopy (TEM) studies in the 1960s and early 1970s using conventional thin section and freeze fracture methodologies revealed ultrastructural bacterial spore appendages. However, the limited technology at that time necessitated the time-consuming process of imaging serial sections and reconstructing each structure. Consequently, the distribution and function of these appendages and their possible role in colonization or pathogenesis remained unknown. By combining high resolution field emission electron microscopy with TEM images of identical bacterial spore preparations, we have been able to obtain images of intact and sectioned Bacillus and Clostridial spores to clearly visualize the appearance, distribution, resistance (to trypsin, chloramphenicol, and heat), and participation of these structures to facilitate attachment of the spores to glass, agar, and human cell substrates. Current user-friendly commercial field emission scanning electron microscopes (FESEMs), permit high resolution imaging, with high brightness guns at lower accelerating voltages for beam sensitive intact biological samples, providing surface images at TEM magnifications for making direct comparisons. For the first time, attachment structures used by pathogenic, environmental, and thermophile bacterial spores could be readily visualized on intact spores to reveal how specific appendages and outer spore coats participated in spore attachment, colonization, and invasion.

Scanning Electron Microscopy Study of Cerebellar Synaptic Junctions

1990 ◽  
Vol 48 (3) ◽  
pp. 148-149
Author(s):  
Orlando J. Castejón
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Cerebellar Cortex ◽  
Salmo Trutta ◽  
Freeze Fracture ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Specimen Preparation ◽  
Scanning Electron

Conventional and high resolution scanning electron microscopy have been applied to trace short cerebellar nerve circuits and to explore the outer and inner surfaces of spine, glomerular and axodendritic synapses. Samples of teleost fishes (Arius spixii and Salmo Trutta) cerebellar cortex were processed according to the slicing technique for conventional scanning electron microscopy (SEM), ethanol cryofracturing technique and freeze-fracture scanning electron microscopy (FFSEM) (Castejón, 1988). Primate (Rhesus monkey) cerebellar cortex was processed for high resolution scanning electron microscopy (Castejón and Apkarian, 1990), according to the protocol of delicate specimen preparation (Peters, 1980). Observations were made in JEOL 100B with (ASID), scanning attachment ISI DS-130 equiped with LaB6 emitter and Hitachi S-900 SEM with a cold cathode field emitter. Micrographs for HRSEM were soft focus printed to reduce instrumental noise (Peters, 1985). A comparison was made of gold-palladium and chromium coating cerebellar samples.The slicing technique and the cryofracture process exposed the neuronal outer surface revealing hidden surface ensheathed by neuroglial cells.

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.

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