Biology Commons^™

A New Approach To Low Temperature Embedding: Quick Freezing, Freeze-Drying And Direct Infiltration In Lowicryl K4m, R. Chiovetti, S. A. Little, J. Brass-Dale, L. J. Mcguffee

Scanning Electron Microscopy

Lowicryl resins are most commonly used for low temperature embedding by progressively lowering the temperature during dehydration. Freeze-substitution has also been successfully used with Lowicryl, but both of these techniques generally rely on chemical fixation and prolonged incubations in organic solvents. Freeze-drying may be combined with embedding in Lowicryl K4M. This technique eliminates all chemical fixation and exposure to organic solvents since the samples are quick-frozen, dried in vacuo and directly infiltrated in pure Lowicryl resin. If a primary aldehyde fixation is desired, freeze-drying may be used as an alternative to dehydration with organic solvents. These new approaches may be …

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 …

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.

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 …

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. …

The Morphology Of Thin Metal Coatings Formed By Rotary Shadowing Biological Species In High Vacuum, J. A. Panitz

Scanning Electron Microscopy

Rotary Shadowing has been used to increase the image contrast of biological species observed during edge-projection imaging in the transmission electron microscope. In this imaging mode, biological species are adsorbed from aqueous solutions onto a highly curved substrate and viewed, over its edge, in a direction parallel to its surface. Since the substrate is not placed between a biological adsorbate and the photographic emulsion that records its image, any material can be used as a support (including high-Z metals and semiconductors). Binding to these technologically interesting materials is observed with unusual clarity and contrast, even at 200kV. Individual adsorbates and …

Plasma Membrane Antigens Detected By Replica Techniques, H. Hohenberg, W. Bohn, G. Rutter, K. Mannweiler

Scanning Electron Microscopy

Methods are introduced for in situ preparation of cell cultures grown on glass coverslips using the replica technique. Special equipment and handling procedures enabled us to prepare large-sized and stable replicas suitable for ultrastructural and immunocytochemical analysis of the different faces of the plasma membrane (PM): the extraplasmic surface (ES), the complementary extraplasmic (EF) and protoplasmic (PF) fracture face, and the protoplasmic surface (PS). Colloidal gold markers in combination with protein A and monospecific/monoclonal antibodies were used to identify virus-specific antigens at the ES of infected cells. Stereo replicas show a coincident location of gold-labeled virus antigens at the ES …

High Resolution Scanning Electron Microscopic Cytology - Specimen Preparation And Intracellular Structures Observed By Scanning Electron Microscopy, T. Inoué

Scanning Electron Microscopy

With the recent developments of the specimen preparation techniques, intracellular organization has been observed three-dimensionally by scanning electron microscopy (SEM). A suitable preparation method is the most important factor for observing intracellular structures at high resolution. Since intracellular structures are usually hidden in the cytoplasmic matrix, SEM observation of them is impossible merely by cracking the fixed cell. To remove the fixed cytoplasm, an osmic maceration technique is the most effective method which is applicable to almost all kinds of specimen preparation. In our laboratory, we developed some methods for observing intracellular structures. Those are the O-D-O method, the A-O-D-O …

Human Metaphase Chromosome Preparation For Scanning Electron Microscopy - A Consideration Of Inherent Problems, T. D. Allen, E. M. Jack, C. J. Harrison, D. Claugher, R. Harris

Scanning Electron Microscopy

Although the physical dimensions of chromosomes are such that they fall well within the spatial resolving power of scanning electron microscopes, results in the past have been disappointing. This is most likely due to limitations in preparative techniques, coupled with the initial necessity to separate the chromosomes from the remainder of the metaphase cell. Two approaches have been employed, a; to use a variety of isolation buffers which provide bulk chromosome preparations, b; to use metaphase spreads prepared essentially as for light microscopy and re-processed for SEM. In the former, wide variations in chromosome surface topography and fibre organisation arise …

Working At Higher Magnifications In Scanning Electron Microscopy With Secondary And Backscattered Electrons On Metal Coated Biological Specimens And Imaging Macromolecular Cell Membrane Structures, Klaus-Ruediger Peters

Scanning Electron Microscopy

Membrane structures of macromolecular dimensions were imaged with high resolution secondary electron type I (SE-I) signal contrasts on metal coated biological specimens. The quality of the surface information was strongly dependent on the signal used for microscopy and on the properties of metal films, i.e., thickness, continuity, structure and decoration effects. Films of 10 nm thickness produced so much type II electrons that identical images were obtained with the conventional SE-II and BSE-II signals. In such images, the type I SE signal was so low that only very weak contrasts were recognizable. If the films - continuous or discontinuous - …

Preparation Of Cryosections For Biological Microanalysis, Karl Zierold

Scanning Electron Microscopy

The element distribution in biological cells and tissues can be revealed by electron probe microanalysis from ultrathin cryosections. In particular, the distribution of physiologically important and often mobile elements such as Na, Mg, P, S, Cl, K, and Ca can be studied in cryosections on an ultrastructural level. The cryopreparation technique required for this purpose consists of 1. cryofixation, 2. cryosectioning, 3. cryotransfer including freeze-drying and carbon coating if necessary, 4. energy dispersive X-ray microanalysis in a cold stage equipped scanning transmission electron microscope. The lateral analytical resolution of this method is less than 50 nm in freeze-dried ultrathin (about …

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 …

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 …

Cryoultramicrotomy And Electron Microanalysis Of Isolated Bovine Adrenal Chromaffin Cells, Richard L. Ornberg

Scanning Electron Microscopy

Instrumentation and techniques have been developed for the cryoultramicrotomy of isolated bovine adrenal chromaffin cells frozen with a liquid helium cooled copper block method. X-ray microanalysis and electron energy loss spectroscopy were used to measure both light and heavy element content of the adrenal storage granule, the chromaffin granule. Ultrathin freeze-dried cryosections usually have a fine compression which in cellular structure compresses the cytoplasm between organelles. Nuclei, chromaffin granules, and vacuoles are relatively free of compression. The mechanism for this distortion is not known al though static charge induced sticking to the knife may be the most probable cause.

The …

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 …

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 …

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 …

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 …