Quantitation in thin Sections Within the Electron Microscope

Author(s):  
J. Edie

In TEM image formation, the observed contrast variations within thin sections result from differential electron scattering within microregions of varying mass thickness. It is possible to utilize these electron scattering properties to obtain objective information regarding various specimen parameters (1, 2, 3).A pragmatic, empirical approach is described which enables a microscopist to perform physical measurements of thickness of thin sections and estimates of local mass, volume, density and, possibly, molecular configurations within thin sections directly in the microscope. A Faraday cage monitors the transmitted electron beam and permits measurements of electron beam intensities.

2001 ◽  
Vol 7 (S2) ◽  
pp. 694-695
Author(s):  
Eric Lifshin ◽  
Raynald Gauvin ◽  
Di Wu

In Castaing’s classic Ph.D. dissertation he described how the limiting value of x-ray spatial resolution for x-ray microanalysis, of about 1 μm, was not imposed by the diameter of the electron beam, but by the size of the region excited inside the specimen. Fifty years later this limit still applies to the majority of measurement made in EMAs and SEMs, even though there is often a need to analyze much finer structures. When high resolution chemical analysis is required, it is generally necessary to prepare thin sections and examine them in an analytical electron microscope where the maximum diameter of the excited volume may be as small as a few nanometers. Since it is not always possible or practical, it is important to determine just what is the best spatial resolution attainable for the examination of polished or “as received” samples with an EMA or SEM and how to achieve it experimentally.


1957 ◽  
Vol 3 (6) ◽  
pp. 1017-1022 ◽  
Author(s):  
Michael L. Watson

Certain phenomena affecting contrast obtained from tissue sections with the electron microscope have been investigated and a technique is described for reducing destruction by the electron beam of fine details in sections. It has been concluded that loss of embedding material is slightly higher at exposed surfaces of sections than it is at surfaces covered by substrate film. Covering of both surfaces of sections with thin films of formvar, collodion, or carbon materially improves the general appearance, reduces distortion, and sometimes reduces loss of tissue mass from the section as result of exposure to the electron beam. This improvement is considered to result from the relatively high melting-point of the covering films which serve to eliminate or reduce surface-tension or other forces operating in methacrylate softened by the electron beam.


Author(s):  
L. D. Jackel

Most production electron beam lithography systems can pattern minimum features a few tenths of a micron across. Linewidth in these systems is usually limited by the quality of the exposing beam and by electron scattering in the resist and substrate. By using a smaller spot along with exposure techniques that minimize scattering and its effects, laboratory e-beam lithography systems can now make features hundredths of a micron wide on standard substrate material. This talk will outline sane of these high- resolution e-beam lithography techniques.We first consider parameters of the exposure process that limit resolution in organic resists. For concreteness suppose that we have a “positive” resist in which exposing electrons break bonds in the resist molecules thus increasing the exposed resist's solubility in a developer. Ihe attainable resolution is obviously limited by the overall width of the exposing beam, but the spatial distribution of the beam intensity, the beam “profile” , also contributes to the resolution. Depending on the local electron dose, more or less resist bonds are broken resulting in slower or faster dissolution in the developer.


Author(s):  
T. J. Beveridge

The Bacillus subtilis cell wall provides a protective sacculus about the vital constituents of the bacterium and consists of a collection of anionic hetero- and homopolymers which are mainly polysaccharidic. We recently demonstrated that unfixed walls were able to trap and retain substantial amounts of metal when suspended in aqueous metal salt solutions. These walls were briefly mixed with low concentration metal solutions (5mM for 10 min at 22°C), were well washed with deionized distilled water, and the quantity of metal uptake (atomic absorption and X-ray fluorescence), the type of staining response (electron scattering profile of thin-sections), and the crystallinity of the deposition product (X-ray diffraction of embedded specimens) determined.Since most biological material possesses little electron scattering ability electron microscopists have been forced to depend on heavy metal impregnation of the specimen before obtaining thin-section data. Our experience with these walls suggested that they may provide a suitable model system with which to study the sites of reaction for this metal deposition.


Author(s):  
E. B. Masurovsky ◽  
H. H. Benitez ◽  
M. R. Murray

Recent light- and electron microscope studies concerned with the effects of D2O on the development of chick sympathetic ganglia in long-term, organized culture revealed the presence of rod-like fibrillar formations, and associated granulofibrillar bodies, in the nuclei of control and deuterated neurons. Similar fibrillar formations have been reported in the nuclei of certain mammalian CNS neurons; however, related granulofibrillar bodies have not been previously described. Both kinds of intranuclear structures are observed in cultures fixed either in veronal acetate-buffered 2%OsO4 (pH 7. 4), or in 3.5% glutaraldehyde followed by post-osmication. Thin sections from such Epon-embedded cultures were stained with ethanolic uranyl acetate and basic lead citrate for viewing in the electron microscope.


Author(s):  
Roberta M. Bruck

An unusual structure in the cochlea is the spiral limbus; this periosteal tissue consists of stellate fibroblasts and collagenous fibers embedded in a translucent ground substance. The collagenous fibers are arranged in vertical columns (the auditory teeth of Haschke). Between the auditory teeth are interdental furrows in which the interdental cells are situated. These epithelial cells supposedly secrete the tectorial membrane.The fine structure of interdental cells in the rat was reported by Iurato (1962). Since the mouse appears to be different, a description of the fine structure of mouse interdental cells' is presented. Young adult C57BL/6J mice were perfused intervascularly with 1% paraformaldehyde/ 1.25% glutaraldehyde in .1M phosphate buffer (pH7.2-7.4). Intact cochlea were decalcified in .1M EDTA by the method of Baird (1967), postosmicated, dehydrated, and embedded in Araldite. Thin sections stained with uranyl acetate and lead citrate were examined in a Phillips EM-200 electron microscope.


Author(s):  
Chester J. Calbick ◽  
Richard E. Hartman

Quantitative studies of the phenomenon associated with reactions induced by the electron beam between specimens and gases present in the electron microscope require precise knowledge and control of the local environment experienced by the portion of the specimen in the electron beam. Because of outgassing phenomena, the environment at the irradiated portion of the specimen is very different from that in any place where gas pressures and compositions can be measured. We have found that differential pumping of the specimen chamber by a 4" Orb-Ion pump, following roughing by a zeolite sorption pump, can produce a specimen-chamber pressure 100- to 1000-fold less than that in the region below the objective lens.


Author(s):  
J. Temple Black

Since its introduction by Fernandez-Moran, the diamond knife has gained wide spread usage as a common material for cutting of thin sections of biological and metallic materials into thin films for examination in the transmission electron microscope. With the development of high voltage E.M. and scanning transmission E.M., microtomy applications will become increasingly important in the preparation of specimens. For those who can afford it, the diamond knife will thus continue to be an important tool to accomplish this effort until a cheaper but equally strong and sharp tool is found to replace the diamond, glass not withstanding.In Figs. 1 thru 3, a first attempt was made to examine the edge of a used (β=45°) diamond knife by means of the scanning electron microscope. Because diamond is conductive, first examination was tried without any coating of the diamond. However, the contamination at the edge caused severe charging during imaging. Next, a thin layer of carbon was deposited but charging was still extensive at high magnification - high voltage settings. Finally, the knife was given a light coating of gold-palladium which eliminated the charging and allowed high magnification micrographs to be made with reasonable resolution.


Author(s):  
R. C. Moretz ◽  
D. F. Parsons

Short lifetime or total absence of electron diffraction of ordered biological specimens is an indication that the specimen undergoes extensive molecular structural damage in the electron microscope. The specimen damage is due to the interaction of the electron beam (40-100 kV) with the specimen and the total removal of water from the structure by vacuum drying. The lower percentage of inelastic scattering at 1 MeV makes it possible to minimize the beam damage to the specimen. The elimination of vacuum drying by modification of the electron microscope is expected to allow more meaningful investigations of biological specimens at 100 kV until 1 MeV electron microscopes become more readily available. One modification, two-film microchambers, has been explored for both biological and non-biological studies.


Author(s):  
R. Sinclair ◽  
B.E. Jacobson

INTRODUCTIONThe prospect of performing chemical analysis of thin specimens at any desired level of resolution is particularly appealing to the materials scientist. Commercial TEM-based systems are now available which virtually provide this capability. The purpose of this contribution is to illustrate its application to problems which would have been intractable until recently, pointing out some current limitations.X-RAY ANALYSISIn an attempt to fabricate superconducting materials with high critical currents and temperature, thin Nb3Sn films have been prepared by electron beam vapor deposition [1]. Fine-grain size material is desirable which may be achieved by codeposition with small amounts of Al2O3 . Figure 1 shows the STEM microstructure, with large (∽ 200 Å dia) voids present at the grain boundaries. Higher quality TEM micrographs (e.g. fig. 2) reveal the presence of small voids within the grains which are absent in pure Nb3Sn prepared under identical conditions. The X-ray spectrum from large (∽ lμ dia) or small (∽100 Ǻ dia) areas within the grains indicates only small amounts of A1 (fig.3).


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