Biology Commons^™

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 …

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 …

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 …