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Detection Of Small (5-15nm) Gold-Labelled Surface Antigens Using Backscattered Electrons, Paul Walther, Martin Müller

Scanning Electron Microscopy

The analysis of surface antigens by scanning electron microscopy represents a practicable alternative to replica techniques. It is generally observed that smaller markers provide for better labelling. In the SEM very small colloidal gold labels however are hardly discerned from contaminants or from surface structures of similar size. They are however unambiguously demonstrated by combining the surface information provided by the secondary electron signal and the material dependent signal provided by the backscattered electrons. A field emission SEM equipped with a highly sensitive single crystal backscattered electron detector allows the routine detection of 5 nm gold particles on biological surfaces. …

Immuno-Cytochemistry With Backscattered Electrons, D. Soligo, E. De Harven, M. T. Nava, G. Lambertenghi-Deliliers

Scanning Electron Microscopy

Some cytochemical reaction products are visible inside the cytoplasm of cells observed with the scanning electron microscope (SEM) using the backscattered electron imaging (BEI) mode. Methods can be utilized whenever they result in the deposition of heavy metal, like silver, lead or osmium at the sites of the enzymatic reaction.

More recently the BEI mode of the SEM has been demonstrated to improve the detection of immunogold labeled cell surface antigens. Colloidal gold particles, 40 to 15 nm in diameter can be efficiently used for immuno-specific labeling. Moreover, cytochemical reactions can be applied to previously immunogold labeled cells, therefore combining …

The State Of Water In The Cell, William Negendank

Scanning Electron Microscopy

A number of scientists in the early 20th century conceived cell water and ions as existing in unique "colloidal" or "bound" states. Work in the 1930's, however, established the assumption that most of cell water and of K exist in a free state like that of a dilute aqueous solution, so that the osmotic activity of cell K serves to balance that of the high concentration of extracellular Na. This assumption underlies theoretical concepts of membrane pumps, Donnan equilibria, and cell potentials, and led to the now classical membrane-osmotic-pump-leak concept of cell ionic distributions and volume maintenance. Nevertheless, the results …

Two Opposing Theories Of The Cell: Experimental Testing By Cryomethods And Electron Microscopy, L. Edelmann

Scanning Electron Microscopy

A main controversial issue in cell biology concerns the molecular mechanism responsible for K⁺ accumulation in living cells and Na⁺ exclusion from them. The alternative theoretical descriptions of these phenomena are based on different assumptions about the physical state of cellular Na⁺, K⁺ and H₂O. In this article it is shown with striated muscles how cryomethods and microanlytical electron microscopy may be used to test the opposing theories. It is concluded that these methods may yield more realistic informations about the physical state of cellular K⁺ and Na⁺ than measurements with …

Demonstrable Fixative Interactions, Keith M. Meek, John A. Chapman

Scanning Electron Microscopy

Electron-optical examination of reconstituted collagen fibrils fixed with tannic acid and/or glutaraldehyde and positively stained with heavy metal anions and cations reveals distinct changes in the high resolution staining patterns seen in TEM. Correlation with the known sequence data demonstrates that these changes are caused by (a) interaction of the fixative with certain charged groups in the collagen and (b) localized stain-exclusion effects following fixation. We have analysed the positive and negative staining patterns from glutaraldehyde-treated collagen in detail. In positive staining, uptake of staining ions is shown to be inhibited not only at residues known to interact with glutaraldehyde …

Tandem Scanning Reflected Light Microscopy, Mojmir Petran, Milan Hadravsky, Alan Boyde, Martin Mueller

Scanning Electron Microscopy

Most attempts at optical microscopy of bulk living objects have failed because of reflections from optical components and the object surface, and overlapping of unsharp out-of-focus images with the one (weak) sharp in-focus image. The optical signal in the image plane thus consists of a strong d.c. component, a weak noise and a still weaker signal. Most of the first two components can be suppressed by scanning in concordance with both the illuminating and the image-forming rays. In the first scan the focussed-on-object plane is illuminated only in several very fine discrete spots. In the second scan only that light …

Cryofixation And Cryosubstitution For Routine Work In Transmission Electron Microscopy, H. Sitte, K. Neumann, L. Edelmann

Scanning Electron Microscopy

After a brief review of the present state of the theory of cryofixation, methods and instruments as well as criteria for the application of cryofixation and cryosubstitution in daily routine work in cell biology and medicine are described. Good results are obtainable using liquid nitrogen for impact freezing on highly polished copper surfaces or by plunging into liquefied propane. Based on these results a versatile and safe system for routine plunging and impact freezing for the majority of biomedical objects has been developed. In order to enable ultramicrotomy at ambient temperature a cryosubstitution system according to the Edelmann principle has …

Problems Associated With The Preparation Of Cell Suspensions For X-Ray Microanalysis Highlighted By The Comparison Of Results With Those Obtained From Tissue Sections, A. Warley, M. D. Kendall, I. W. Morris

Scanning Electron Microscopy

Fully quantitative X-ray microanalysis of freeze dried frozen sections allowed the concentration of monovalent ions in the nuclei of mouse thymocytes in tissue sections to be compared with those from thymocytes which had been isolated by a simple preparation procedure. Isolated cells showed increased concentrations of Na and Cl. This was further investigated in the rat where the size of the thymus a 11 owed comparison between different isolation media using cells derived from the same animal. Use of autologous serum as the final suspending medium gave reproducible results, whereas with suspension in BSS, or 20% dextran the results were …

Low Temperature Embedding, E. Carlemalm, W. Villiger, J. -D. Acetarin, E. Kellenberger

Scanning Electron Microscopy

The Lowicryl resins K4M and HM20 are methacrylate/acrylate based formulations which can re used for embedding biological material at low temperature in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. The resins are applicable over a very extended temperature range, approximately 210°K to 340°K. Even lower temperatures down to ca. 190°K can be reached with two new resins, K11M and HM23. Test embeddings of unfixed material after freeze-substitution have given promising results which could re useful for imnunocytochemical labeling. Lipid extraction is small or absent when the two new resins are used in combination with …

Cryopreparation Of Tissue For Electron Microscopy, John G. Linner, Stephen C. Bennett, Donna S. Harrison, Alton L. Steiner

Scanning Electron Microscopy

An apparatus able to remove amorphous phase tissue water without recrystallization or rehydration has been produced. Application of this technique to biological samples achieves both the preservation of ultrastructure and the retention of cellular macromolecules and solute without redistribution or modification.

Small pieces of fresh tissue were cryofixed by the method of bounce free metal-mirror freezing on polished copper bars at liquid nitrogen temperature. Tissue samples were then placed under liquid nitrogen in a copper sample holder equipped with a thermocouple and feedback controlled heating circuit. Under liquid nitrogen the sample block was placed in a stainless steel sample chamber …

Freeze Substitution And Low Temperature Embedding, B. Humbel, M. Müller

Scanning Electron Microscopy

The problems of conventional EM-preparation techniques based on chemical fixation may be overcome to a considerable extent by freeze substitution techniques. Although at present substitution cannot be performed at sufficiently low temperatures to prevent the recrystallization of vitrified aqueous specimens, thin sectioned biological samples show an improved information density. If freeze-substitution is combined with conventional embedding above 273 K (Epon/Araldite, Spurr's resin) the substituting organic solvent must contain stabilizing agents such as osmiumtetroxide, glutaraldehyde or uranylions. In combination with low temperature embedding procedures (Lowicryl) completely unfixed samples are obtained, which are suitable for immunolabelling and electron spectroscopic experiments. Water in …

Colloidal Gold Particles Detected On Highly Structured Surfaces Of Large Samples By Backscattered Electrons In The Scanning Electron Microscope, D. Studer, R. Hermann

Scanning Electron Microscopy

Colloidal gold labelled surface antigens on large, highly structured samples were unambiguously visualized at high magnification with backscattered electrons at 25 kV in a field emission scanning electron microscope (SEM). Secondary electron (SE) and backscattered electron (BSE) images of large and highly structured specimens are frequently disturbed by charging effects. By optimizing the thickness of the conducting carbon layer and the intensity of the incident electron beam, this problem can be overcome. The midgut of the lepidopteran larvae of Spodoptera litoralis was used as specimen. The activated 𝛿-endotoxin of Bacillus thuringiensis coupled to colloidal gold binds to the microvilli exposed …

Immunocytochemical Labeling Of Enzymes In Low Temperature Embedded Plant Tissue: The Precursor Of Glyoxysomal Malate Dehydrogenase Is Located In The Cytosol Of Watermelon Cotyledon Cells, C. Sautter

Scanning Electron Microscopy

The Lowicryl-technique in combination with protein A gold was used in order to localize the precursor of glyoxysomal malate dehydrogenase in watermelon cotyledons. Preservation of the antigen was evaluated by a preembedding technique in isolated organelles. The glyoxysomal malate dehydrogenase was localized in tissue sections by a postembedding technique. Antigens of glyoxysomal malate dehydrogenase were found in the glyoxysomal matrix and in the cytosol, whereas the endoplasmic reticulum was completely free of labeling. Controls are presented by preimmunserum, by a serum against various proteins of the glyoxysomal membrane and by application of cycloheximide in order to inhibit translation at cytosolic …

To Resin Or Not To Resin In Immunocytochemistry, J. J. Wolosewick

Scanning Electron Microscopy

Hydrated resinless sections produced by a variety of methods (cryoultramicrotomy or polyethylene glycol techniques) appear to be excellent specimens for post-embedding immunocytochemistry at the electron microscopic level. A perplexing problem is the lack of apparent penetration throuqhout the section thickness of particulate probes such as ferritin or colloidal gold. This report draws attention to the possible causes for this phenomenon namely the size of the probe and the possible effects of fixation or processing. Smaller probes, gentler fixation or permeabilization procedures and increased incubation times all seem to be logical approaches to increasing penetration of immunoreagents into thick hydrated sections.

X-Ray Microanalysis In Cryosections Of Natively Frozen Paramecium Caudatum With Regard To Ion Distribution In Ciliates, M. Schmitz, R. Meyer, K. Zierold

Scanning Electron Microscopy

Cells of Paramecium caudatum were shock-frozen without pretreatment for cryoultramicrotomy and freeze-dried for subsequent X-ray microanalysis. Na, Mg, P, S, Cl, K, and Ca were detected in different amounts in several subcellular compartments. In particular, calcium was localized below the cell surface (pellicle). Trichocysts were found to contain significant amounts of Na in their base but not in the tip. Na, Mg, P, S, Cl, K, Ca were found in electron dense deposits within the lumen of the contractile vacuole. A small K concentration was found in the cytoplasm and in the mitochondria. X-ray microanalysis of the element distribution in …

Scanning Electron Microscopy In Retinal Research, Bessie Borwein

Scanning Electron Microscopy

The literature on scanning electron microscopy (SEM) pertaining to the retina has been surveyed and described.

The first two papers on SEM and retina appeared in 1969. Most of the earlier studies concentrated on descriptions [by SEM alone, or with light microscopy (LM) and transmission electron microscopy (TEM)] of the appearance of various retinae and retinal cells [fish, newt, primates, rodents and rabbits, bullfrog]; or embryology of the chick retina. Two papers dealt with retinal disease. In all there were 25 papers in SEM/retinal research before and in 1974. Since 1975 there have been 111 papers which have used SEM …

A Rapid Technique For Counting Cracks In Rocks, W. B. Durham, J. M. Beiriger, H. C. Weed

Scanning Electron Microscopy

Using a scanning electron microscope (SEM) and an image analyzer, we have developed a technique for counting and measuring cracks in rocks which is more efficient than traditional techniques in which an operator performs all image analysis functions. The key aspect of the technique is that black-on-white tracings of fresh cracks, which can be made rather rapidly by an operator, are measured and digitized by an image analyzer. The most time-consuming step in the process has now become the generation of SEM micrographs and pertinent chemical (mineralogical) information, not the quantification of crack structure. The technique has been applied to …

Surface Roughness Contribution To The Auger Electron Emission, D. Wehbi, C. Roques-Carmes

Scanning Electron Microscopy

Scanning Auger Microscopy (SAM) experiments have shown that z height and θ slope relative to the analysed spot are parameters that contribute to the measured Auger intensity I(z, θ). For greater analysed areas specific to Auger Electron Spectroscopy (AES), the knowledge of height and slope statistical distributions P(z) and P(θ) is required. These functions have been determined by means of profilometric data. The spatial resolution of the used tactile profilometer is similar to that which characterizes AES. A mathematical relationship I { P(z), P(θ) } has been set up for Si samples whose roughness is well defined. On the other …

Sputtered Thermal Ion Mass Spectrometry As A New Quantitative Method For In-Depth Analysis, G. Blaise

Scanning Electron Microscopy

The Sputtered Thermal Ion Mass Spectrometry method (STIMS) consists of collecting a part of the matter sputtered from a solid by ion bombardment into a heated cell where it is reduced into atoms. A thermal ionization process or an electron impact process taking place in the cell yields ions which are extracted and mass analyzed. The composition of the solid is determined from ion intensities after calibration of ionization coefficients. It has been demonstrated that the method has an absolute quantitative character. Applications to elemental quantitative analysis (identification of new compounds in diffusion couples, dust particle analysis) and in-depth analysis …

Wave-Length Dispersive Microprobe Analysis Of Coated Samples Of Bulk Tissues, C. Ward Kischer, Thomas M. Teska

Scanning Electron Microscopy

Hypertrophic scars contain highly pleomorphic cells, including many from the erythrocytic series which have been extravasated. The conventional visual mode of SEM cannot distinguish the cell types with certainty except in the case of typical biconcave disc-shaped erythrocytes. Microprobe elemental analysis might be used to differentiate one type from another on the basis of iron and possibly phosphorus (for nucleated cells). Using coated specimens (gold or gold-palladium) precludes simultaneous visual mode SEM with EDX because of energy line interference with phosphorus and other elements. However, wave-length dispersive analysis offers minimal or no interference, and a coated specimen offers the use …

Progress In Element Analysis On A High-Voltage Electron Microscope, W. F. Tivol, D. Barnard, T. Guha

Scanning Electron Microscopy

X-Ray microprobe (XMA) and electron energy-loss (EELS) spectrometers have been installed on the high-voltage electron microscope (HVEM). The probe size has been measured and background reduction is in progress for XMA and EELS as are improvements in electron optics for EELS and sensitivity measurements.

XMA is currently useful for qualitative analysis and has been used by several investigators from our laboratory and outside laboratories. However, EELS background levels are still too high for meaningful results to be obtained. Standards suitable for biological specimens are being measured, and a library for quantitative analysis is being compiled.

Oxidation Of Aluminum Studied By Ion Scattering Spectroscopy (I.S.S) In A Scanning Auger Microscope, M. Gautier, J. P. Duraud, J. P. Vigouroux, C. Le Gressus, R. Shimizu

Scanning Electron Microscopy

The set up of an ion gun, producing a focused beam in the analysis chamber of a Scanning Auger Microscope permits ion scattering experiments: surface studies performed by electron spectroscopies can then be enlarged by Ion Scattering Spectroscopy (I.S.S.) to get additional information.

I.S.S. appears to be very sensitive to the cleanliness of the surface: comparison between Electron Energy Loss Spectroscopy (E.E.L.S.) and I.S.S. studies on clean samples show that I.S.S. can still detect oxygen even when it is not detectable by E.E.L.S.

Preliminary results on oxidation of Al (111) and Al (100) give oxidation curves in good agreement with …

Cross-Sectional Analysis Of Silicon Metal Oxide Semiconductor Devices Using The Scanning Electron Microscope, Daniel S. Koellen, David I. Saxon, Kenneth E. Wendel

Scanning Electron Microscopy

A technique has been developed which enables one to cross-section specific devices or features for examination with the scanning electron microscope (SEM). This method is used for investigation of all facets of microelectronic circuit manufacture from research and development to failure analysis of the finished product.

Selective etching is used to provide contrast to each processed layer. Etch type and sequence, used for delineation, are important to understand since they may add artifacts to the cross-section, leading to erroneous analysis conclusions. The etchant and etch conditions used will be dictated by the information needed from a particular sample.

Etching systems …

Digital Processing Of Electron Energy Loss Spectra And Images, R. D. Leapman, K. E. Gorlen, C. R. Swyt

Scanning Electron Microscopy

Processing in electron energy loss spectroscopy involves both data acquisition and analysis. The interface of an analytical electron microscope to a laboratory computer with a satellite microcomputer dedicated to data acquisition results in a system with a high degree of flexibility. In spectrum acquisition, channels may be selected around specific core edges, or dwell times may be varied continuously as a function of energy loss to reduce the dynamic range of the signal. Data transfer to the host computer allows further analysis such as the removal of plural scattering by spectral deconvolution. Elemental maps and line-scans can be recorded with …

Intracellular Localization Of Heavy Metals In Yeast By X-Ray Microanalysis, Ljerka Kunst, Godfried M. Roomans

Scanning Electron Microscopy

The intracellular localization of heavy metals in yeast cells was studied by means of energy-dispersive X-ray microanalysis. The yeast, Saccharomyces cerevisiae, was pretreated with phosphate and then loaded with different metal ions, by suspending the cells in salt solutions (Ni, Zn, Cd, Pb, Al and Cr). For the analysis, the cells were embedded in gelatin, rapidly frozen, and thin cryosections were cut on a dry knife.

A considerable uptake of divalent cations by the yeast cells was found to occur. The cations were bound to the polyphosphate granules localized in or close to the cell vacuoles. Immediately after phosphate …

Alterations Of The Intracellular Water And Ion Concentrations In Brain And Liver Cells During Aging As Revealed By Energy Dispersive X-Ray Microanalysis Of Bulk Specimens, Gy. Lustyik, I. Zs.-Nagy

Scanning Electron Microscopy

Age dependence of the intracellular concentrations of monovalent ions (Na+, K+ and Cl-) was examined in 1, 11 and 25-month-old rat brain and liver cells by using energy dispersive X-ray microanalysis. The in vivo concentrations of Na+, K+ and Cl- ions were calculated from two different measurements: The elemental concentrations were measured in freeze-dried tissue pieces, and the intracellular water content was determined by means of a recently developed X-ray microanalytic method, using frozen-hydrated and fractured bulk specimens as well as subsequent freeze-drying.

All the single monovalent ion concentrations and consequently, also the total monovalent ion content showed statistically significant …

Study By Scanning Electron Microscopy And Electron Spectroscopy Of The Cascade Of Electron Multiplication In An Insulator Submitted To An Electric Field, J. P. Vigouroux, J. P. Duraud, C. Le Gressus, G. Petite, P. Agostini, C. Boiziau

Scanning Electron Microscopy

An original method for revealing the dielectric heterogeneities on an insulating surface has been developed on creation of an electron multiplication cascade inside the insulator placed in an electric field. The steps of the physical process are: (i) excitation of electrons into the conduction band, (ii) electric field acceleration of the conduction electrons, (iii) ionization of the valence levels, (iv) creation of many more new defects in the vicinity of dielectric heterogeneities, (v) charge localization on defects and appearance of a local residual potential. The potential map is observable by scanning electron microscopy after propagation of the ionizing cascade, but …

High Accuracy And Automatic Measurement Of The Pattern Linewidth On Very Large Scale Integrated Circuits, H. Yamaji, M. Miyoshi, M. Kano, K. Okumura

Scanning Electron Microscopy

High accuracy measurement of pattern linewidth is particularly important in Very Large Scale Integrated Circuits (VLSI) manufacturing.

The measurement of pattern linewidth has been done by optical methods. However, the optical methods have several problems: as the measured value depends on slope angle at pattern edge, thickness and optical property of film and also substrate, there exists a large difference in size (0.3 μm) between the defined edge and the true edge in case of photoresist linewidth measurements. Especially, the optical methods have severe problems to measure bottom of pattern edge and are unsuitable to measure pattern linewidth in VLSI's …

A Wien Filter Energy Loss Spectrometer For The Dedicated Scanning Transmission Electron Microscope, P. E. Batson

Scanning Electron Microscopy

A Wien filter electron spectrometer has been added to a VG Microscopes, Ltd. HB501 STEM to improve the energy resolution and accuracy of energy loss analysis combined with a high spatial resolution. An energy resolution of 130meV is obtained with a 2mR collection angle at the specimen. The 0.28eV wide field emission energy profile therefore dominates the energy resolution for the device. The energy axis is automatically calibrated by the electrostatic method of scanning, yielding an accuracy and stability of 30meV. A preliminary energy resolution of 0.5eV is demonstrated for 20mR full collection angles at the specimen. Results of experiments …

Scanning Electron Microscopy Of The Lateral Ventricle Of The Pigeon Brain, P. Mestres, K. Rascher, J. D. Delius

Scanning Electron Microscopy

Adult pigeons of both sexes were used for this study. Depending upon the distribution of various surface profiles, for example cilia, microvilli and blebs, ependymal areas with differing surface patterns were distinguished in the lateral ventricle. The topographical locations of these areas with respect to the underlying forebrain nuclei were determined in accord with the atlas of Karten and Hodos (1967). The medial surface (A) of the ventricle was much more densely ciliated than the lateral surface (B). There did not appear to be any correlation between a given surface pattern and a specific type of underlying nervous tissue. Comparison …