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Freeze-Drying And Related Preparation Techniques For Biological Microprobe Analysis, Romuald Wróblewski, Joanna Wróblewski, Matti Anniko, Lars Edström

Scanning Electron Microscopy

An X-ray microanalytical and morphological investigation has been carried out on rapidly frozen, freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerisation procedures following freeze-substitution and freeze-drying. The investigation also included an analysis of specimens infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl HM20. The morphological preservation of Lowicryl embedded tissue was adequate for the identification of different cell structures like nuclei, mitochondria, lysosomes and different types of endoplasmic reticulum. X-ray microanalytical investigation of low temperature embedded material displayed an elemental composition of cells and organelles similar to that found in freeze-dried …

Recent Developments In Electrical Microcharacterization Using The Charge Collection Mode Of The Scanning Electron Microscope, D. B. Holt, M. Lesniak

Scanning Electron Microscopy

There are six distinguishable types of charge collection (CC) signal. Consequently specially designed CC detection systems are needed to obtain e.g. true EBIC and EBIV measurements and make quantitative electrical microcharacterization possible. EBIC and EBIV can arise from fields due to barriers such as p-n junctions or due to changes in doping e.g. p to p+. Materials, defect and device parameters are calculable from these signals. Hot-cold stages are important for temperature dependence studies of contrast, for improving signal to noise ratios and reducing leakage currents. Image processing and pattern recognition methods are vital for rapidly locating and evaluating the …

Applications Of Scanning Electron Microscopy And X-Ray Microanalysis In Inner Ear Pathology, M. Anniko, D. J. Lim, A. Sobin, R. Wróblewski

Scanning Electron Microscopy

Surface pathology of inner ear structures so far described in detail concern cochlear and vestibular hair cells and the stria vascularis. In man, surgical intervention into the inner ear is very uncommon and when performed is in general with the primary objective of destroying the diseased peripheral end organs. The vast majority of inner ear tissue available for use with scanning electron microscopy (SEM) is therefore obtained from animals.

The present paper reviews the progression of surface pathology caused by aminoglycoside antibiotics, acoustic overstimulation and in a guinea pig strain with genetic inner ear disease. The primary site of onset …

Cell Movement And Contraction In Somite Development, Ellen A. G. Chernoff

Scanning Electron Microscopy

During somite formation the segmental plate mesoderm, lying on either side of the axial organs, reorganizes into roughly spherical pairs of epithelial structures. This segmentation process includes changes in cell shape and position, cell-cell and cell-substratum adhesive properties and accumulation of extracellular matrix material which proceed down the anterior-posterior axis. Later in somite development the sclerotome region "disperses", migrating around the spinal cord where it produces the cartilage model of the vertebral column. Experimental manipulation of segmentation and sclerotome dispersal with drugs affecting microfilaments, microtubules and calcium-dependent contraction suggest that cells migrate into position, elongate, and undergo apical contraction as …

Ultrastructural Studies Of Crystal-Organic Matrix Relations In Renal Stones, P. -T. Cheng, A. D. Reid, K. P. H. Pritzker

Scanning Electron Microscopy

Biochemically the organic matrix of kidney stones contains mucoproteins, mucopolysaccharides, inorganic material and bound water. Morphologically, the organic matrix exists as either amorphous or fibrous forms. We have attempted to critically evaluate results from analytical and morphological studies on stone matrices using light microscopy, histochemistry, x-ray diffraction, scanning electron microscopy, x-ray energy dispersive spectrometry, transmission electron microscopy and selected area electron diffraction.

On the surface of calcium oxalate stones, there are usually large masses of randomly deposited calcium oxalate crystals each coated with organic matrix. Transmission electron microscopy shows these large surface crystals are composed of rows of smaller crystallites …

Electron Beam Induced Damage On Passivated Metal Oxide Semiconductor Devices, S. Görlich, E. Kubalek

Scanning Electron Microscopy

Electron beam testing of integrated circuits (IC) is currently based on the electron beam induced conductivity in insulators to short the passivation layer and to enable a voltage measurement at covered conductor tracks. However, applying this technique to passivated MOS devices causes severe radiation damage, which was at first explained by primary electrons penetrating into the deep-lying gate oxide. Nondestructive electron beam testing was expected by using low electron energies that do not allow the primary electrons to reach into the gate oxide.

Therefore here the influence of nonpenetrating electron irradiation on the characteristics of passivated NMOS transistors has been …

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 …

In Vivo Light Microscopy Of Organs, Robert S. Mccuskey

Scanning Electron Microscopy

High resolution, brightfield and fluorescence, light microscopic methods have been developed for examining living organs in situ. The methods permit study of the rate, duration, magnitude and direction of dynamic histologic, pathologic, physiologic and pharmacologic events. In addition, morphometric analyses of such living preparations can provide basic information needed to evaluate alterations induced by fixation and processing of these organs for electron microscopy. Most organs are amenable to such investigations. In anesthetized animals, the selected organ is trans-and/or epi-illuminated with selected wavelengths of monochromatic light, imaged with water immersion objectives and the resulting monochromatic optical images televised using silicon …

Physics Of The Preparation And Observation Of Specimens That Involve Cryoprocedures, E. Kellenberger, E. Carlemalm, W. Villiger

Scanning Electron Microscopy

With this introductory chapter we attempt a synthesis of old and new knowledge of the physical principles that govern cryomethods. Interface phenomena determine the increase or decrease of the number of particles observed in frozen-hydrated suspensions because they occupy the air-liquid interface according to their specific balance of hydrophobic versus hydrophilic properties. Biological macromolecules are surrounded by organised water, the hydration shell, that prevents them from sticking to each other. Partial or complete removal of these hydration shells by freeze-drying or freeze-substitution leads to collapses or aggregations. The solvent-induced aggregation is usually decreased by prior cross-linking with adequate chemical fixatives. …

On The Application And Possibilities Of In Vivo Microscopy In Liver Research, E. Wisse, R. S. Mccuskey

Scanning Electron Microscopy

In vivo microscopy (IVM) provides a valuable method for studying the histophysiology of the living liver. The method allows observation of living cells in the intact organ of an anesthetized animal with an undisturbed microcirculation, at a magnification and a resolution comparable to normal light microscopy of sectioned material. Due to the absence of preparative procedures, the image differs substantially from histological sections, but it has the advantage of providing us with a reference preparation free of artifacts. In the case of the liver, we have the opportunity to observe directly such details as bile capillaries, intracellular fat droplets, lysosomes, …

Glutaraldehyde Fixation Chemistry A Scheme For Rapid Crosslinking And Evidence For Rapid Oxygen Consumption, Timothy J. A. Johnson

Scanning Electron Microscopy

The chemical literature suggests that pyridine derivatives are the major reaction products of amine-aldehyde reactions. A scheme is proposed for pyridine synthesis which is derived from a classic synthesis of pyridines from one amine molecule and 3 aldehyde molecules. The pyridine polymers are considered as a major class of cross-links in glutaraldehyde fixed cells. This scheme for pyridine synthesis accounts for the following observations of amine-glutaraldehyde react ions: 1) large quantities of acid are rapidly produced, 2) oxygen uptake occurs at a high rate, 3) a variety of products distinguished by molecular size are rapidly and irreversibly synthesized, and 4) …

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 …

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 …

Effects Of Temperature And Crowding On The Pathogenicity Of Edwardsiella Ictaluri In Channel Catfish (Ictalurus Punctatus), Sharon L. Johnson, Lawrence W. Hinck

Journal of the Arkansas Academy of Science

Channel catfish were injected with Edwardsiella ictaluri and stocked at increasing temperatures and densities. Bacteriological examination of kidney, liver and spleen revealed the greatest numbers of organisms in fish from the highest temperature and stocking density tested. Survival time was the shortest for fish held at the highest temperature and stocking density. Increased temperature and crowding were directly proportional to the number of organisms recovered from the organs and inversely proportional to fish survival time.

Cyperus Flavicomus, The Correct Name For Cyperus Albomarginatus, Gordon C. Tucker

Faculty Research & Creative Activity

The correct name for Cyperus albomarginatus, a pantropical and warm-temperate sedge, is shown to be C. jlavicomus Michaux.

Cyperus Flavicomus, The Correct Name For Cyperus Albomarginatus, Gordon Tucker

Faculty Research & Creative Activity

The correct name for Cyperus albomarginatus, a pantropical and warm-temperate sedge, is shown to be C. jlavicomus Michaux.

Biotic Interactions Among Estuarine Infaunal Opportunistic Species, Roman Zajac, R. B. Whitlatch

Biology and Environmental Science Faculty Publications

Biotic interactions among soft-sediment infauna were investigated in a small New England estuary in order to determine what effect(s) established opportunistic species had on subsequent recolonization. Interactions were defined according to successional models developed by Connell and Slatyer (1977), e.g. facilitation, tolerance and inhibition. Adults of the opportunistic polychaetes Streblospio benedicti, Polydora ligni and Hobsonia florida were added at 2 densities to separate cores containing defaunated sediment. These cores and control cores containing no worms were sampled at 10 d intervals for 40 d. Cores containing capillary tubes to simulate polychaete tubes were also deployed and sampled at 10 d …

The Quaternary Bone Caves And Associated Sites At Wallingford, Jamaica, Donald A. Mcfarlane, R. E. Gledhill

WM Keck Science Faculty Papers

A group of caves associated with the sink of the One Eye River in St. Elizabeth Parish, Jamaica, have been the subject of numerous important palaeontological investigations beginning 1919. Unfortunately, considerable confusion has arisen in the literature through inadequate documentation of different sites. The caves of the immediate area are described and located, and their palaeontological significance is summarised in the light of recent taxonomic review and relevant geochronological evidence.

Concanavalin A-Binding Enzymes Of Crotalus Scutulatus Scutulatus Venom, Michael W. Hinson, C. K. Childs, Bob D. Johnson, Dewey H. Sifford

Journal of the Arkansas Academy of Science

Crotalus scutulatus scutulatus crude venom was separated into two fractions by Concanavalin A Sepharose 4B affinity chromatography. The proteins binding to Con A exhibited phosphomonoesterase (orthophosphoric monoester phosphohydrolase EC 3.1.3.2), phosphodiesterase, 5'-nucleotidase (5'-ribonucleotide phosphohydrolase EC 3.1.3.5), phospholipase A(phosphatidate 2-acylhydrolase EC 3.1.1 .4), hyaluronidase (hyaluronate glycanohydrolase EC 3.2.1 d), N-benzoyl-L-arginine ethyl esterase, p-toluenesulfonyl-L-arginine methyl esterase, L-amino acid oxidase (L-amino acid: 02 oxidoreductase [deaminating] EC 1.4.3.2), and caseinolytic activities. Thrombin-like and NAD nucleosidase (5'-ribonucleotide phosphohydrolase EC 3.1.3.5) activities were not observed. The crude venom and the fraction containing the glycoproteins which bound to Con A were fractionated by DEAE Sephadex A-50 ion …

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 …

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 …

Observation Of Colloidal Gold Labelled Platelet Surface Receptors And The Underlying Cytoskeleton Using High Voltage Electron Microscopy And Scanning Electron Microscopy, R. M. Albrecht, J. A. Oliver, J. C. Loftus

Scanning Electron Microscopy

Fibrinogen conjugated to colloidal gold or colloidal gold-monoclonal anti-glycoprotein IIb/IIIa (fibrinogen receptor) was used to label the receptor on platelets. Whole mount preparations were examined by stereo pair high voltage electron microscopy and then by scanning electron microscopy to determine the feasibility of this approach in detecting the number of receptors and their location relative to the cytoskeletal and surface structure. Both the ligand-gold and antibody-gold labels were effective. The relative numbers of receptors could be seen and their relationship to cytoskeletal structure could be determined. Marked differences in receptor number and distribution were observed when platelets in different stages …

Cell Volume Regulation Studies With The Electron Microprobe, Mary Jo Ingram, F. Duane Ingram

Scanning Electron Microscopy

Lumbricals from the hind feet of young rats are dissected free, stretched to approximately 125% of resting length, and mounted on individual, simple plastic forms. After recovery in physiological saline, the isolated muscles are incubated for periods of 40 to 60 min. in one of a series of hypertonic bathing solutions. The composition of each bathing solution is identical, except for osmolality which is increased with lactose. At least one muscle from each animal is incubated in a control solution to serve as a control muscle for that particular set of 5 to 8 muscles. Mounted muscles are removed from …

Does The Embedding Chemistry Interact With Tissue?, B. E. Causton

Scanning Electron Microscopy

Resins and resin compounds interact with tissue in two ways, physico-chemically and by chemical reaction. The physico-chemical influences affect both the structure and biological activity of the tissue causing proteins to change shape or phase equilibria to be disrupted by a change in solvent or by the growth of polymer networks within existing biological polymeric structures. Chemical reactivity between tissue and resin components also reduces biological activity by changing either the structure of polymeric matter already present, e.g., crosslinking proteins, grafting hydrophobic resins onto hydrophilic protein backbones, or by modifying the hydrophobicity of specific sites, e.g., acylation of amino groups …

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 …

Complementary Replicas Of Ultra-Rapidly Frozen Specimens, M. J. Costello, R. D. Fetter, T. G. Frey

Scanning Electron Microscopy

Complementary freeze-fracture replicas have been prepared of ultra-rapidly cooled specimens in the absence of chemical pretreatments. The grid-sized replicas were stabilized by open mesh gold grids during the cleaning process and, after cleaning, were supported on thin Formvar films. The complementary replicas were valuable for describing artifacts, for interpreting the nature of fracture planes and for evaluating the resolution of replicas. Complementary images demonstrated that heat emitted from resistance electrodes or electron guns during evaporation can seriously distort fracture surfaces even for samples held at -150°C. Complementary images of crystalline membranous cytochrome c oxidase helped establish that fracturing reveals hydrophilic …

The Glass Knife In Ultramicrotomy, Lahja Sevéus, Cecilia Tarras-Wahlberg

Scanning Electron Microscopy

To obtain the best ultrathin sections in ultramicrotomy and cryoultramicrotomy, a high quality knife is of paramount importance. Glass knives were introduced for ultramicrotomy, in the manually broken form, in 1950. Since the introduction of mechanical knifemakers, the production of glass knives has become a standardized laboratory routine. In conventional ultramicrotomy different knife angles are used to cover different specimens and embedding media. The 50° knife broken with a small deviation of the score from the diagonal of the square gives a long useful edge, especially if damping during breaking is used. The 50° knife is, therefore recommended as the …

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 …

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 …