Ultrathin in‐plane heterostructures for efficient CO2 chemical fixation

Osmoregulatory mechanisms in algae were investigated mainly from a physiological point of view (KAUSS 1977, HELLEBUST 1976). In Porphyra two osmotic agents, i. e. floridoside/isofloridoside (KAUSS 1968) and certain ions, such as K+ and Na+(EPPLEY et al. 1960) are considered for osmotic balance. Accumulations of ions (particularly Na+) in the cytoplasm during osmotic adaptation is improbable, because the activity of enzymes is generally inhibited by high ionic concentrations (FLOWERS et al. 1977).The cellular organization of Porphyra was studied with special emphasis on the development of the vacuolar system under different hyperosmotic conditions. Porphyra was cultivated at various strengths of the culture medium ASP 12 (PROVASOLI 1961) ranging from normal to 6 times concentrated (6x) culture medium. Por electron microscopy freeze fracturing was used (specimens fixed in 2% glutaraldehyde and incubated in 30% glycerol, preparation in a BALZERS BA 360 M apparatus), because chemical fixation gave poor results.

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Artifacts caused by dehydration and epoxy embedding in TEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008599x ◽

1991 ◽

Vol 49 ◽

pp. 338-339

Author(s):

Hilton H. Mollenhauer

Keyword(s):

Electron Microscopy ◽

Electron Beam ◽

Volume Change ◽

Tissue Damage ◽

Beam Specimen ◽

Chemical Fixation ◽

Resin Impregnation ◽

Storage Vesicles ◽

A Cell ◽

Tissue Shrinkage

Various means have been devised to preserve biological specimens for electron microscopy, the most common being chemical fixation followed by dehydration and resin impregnation. It is intuitive, and has been amply demonstrated, that these manipulations lead to aberrations of many tissue elements. This report deals with three parts of this problem: specimen dehydration, epoxy embedding resins, and electron beam-specimen interactions. However, because of limited space, only a few points can be summarized.Dehydration: Tissue damage, or at least some molecular transitions within the tissue, must occur during passage of a cell or tissue to a nonaqueous state. Most obvious, perhaps, is a loss of lipid, both that which is in the form of storage vesicles and that associated with tissue elements, particularly membranes. Loss of water during dehydration may also lead to tissue shrinkage of 5-70% (volume change) depending on the tissue and dehydrating agent.

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Improvement of an artificial test substrate technique for evaluation of em immunocytochemical labeling

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128122 ◽

1987 ◽

Vol 45 ◽

pp. 762-763

Author(s):

G. D. Gagne ◽

M. F. Miller

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Bovine Serum ◽

Artificial Substrate ◽

Chemical Fixation ◽

As If

We recently described an artificial substrate system which could be used to optimize labeling parameters in EM immunocytochemistry (ICC). The system utilizes blocks of glutaraldehyde polymerized bovine serum albumin (BSA) into which an antigen is incorporated by a soaking procedure. The resulting antigen impregnated blocks can then be fixed and embedded as if they are pieces of tissue and the effects of fixation, embedding and other parameters on the ability of incorporated antigen to be immunocyto-chemically labeled can then be assessed. In developing this system further, we discovered that the BSA substrate can also be dried and then sectioned for immunolabeling with or without prior chemical fixation and without exposing the antigen to embedding reagents. The effects of fixation and embedding protocols can thus be evaluated separately.

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Observations of thick and thin sections in a field-emission SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100172826 ◽

1994 ◽

Vol 52 ◽

pp. 1018-1019

Author(s):

W. P. Wergin ◽

S. Roy ◽

E. F. Erbe ◽

C. A. Murphy ◽

C. D. Pooley

Keyword(s):

Electron Microscope ◽

Field Emission ◽

Room Temperature ◽

Osmium Tetroxide ◽

Uranyl Acetate ◽

Chemical Fixation ◽

Thin Sections ◽

Transmission Electron ◽

Conventional Transmission Electron Microscope ◽

Scanning Electron

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freezesubstituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures. Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's medium and sectioned for observation with a Hitachi S-4100 field emission scanning electron microscope that was equipped with an Oxford CT 1500 Cryotrans System. Thin sections, about 80 nm thick, similar to those generally used in conventional transmission electron microscope (TEM) studies were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min. Sections about 2 μm thick were also mounted and stained in a similar fashion. The grids were mounted on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at ambient temperature. Thick and thin sections of the larvae were evaluated and photographed in the SEM at different accelerating voltages. Figs. 4 and 5 have undergone contrast conversion so that the images would resemble transmitted electron micrographs obtained with a TEM.

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Freeze substitution fixation of fungal reproductive structures and spores

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156936 ◽

1989 ◽

Vol 47 ◽

pp. 990-991

Author(s):

C. W. Mims ◽

E. A. Richardson

Keyword(s):

Plant Pathogens ◽

Freeze Substitution ◽

Uranyl Acetate ◽

Chemical Fixation ◽

Dialysis Membrane ◽

Razor Blade ◽

Thin Sections ◽

Reproductive Structures ◽

Dry Down ◽

Diamond Knife

The advantages of freeze substitution fixation over conventional chemical fixation for preservation of ultrastructural details in fungi have been discussed by various authors. As most ascomycetes, basidiomycetes and deuteromycetes do not fix well using conventional chemical fixation protocols, freeze substitution has attracted the attention of many individuals interested in fungal ultrastructure. Thus far most workers using this technique on fungi have concentrated on thin walled somatic hyphae. However, in our laboratory we have experimented with the use of freeze substitution on a variety of fungal reproductive structures and spores with promising results.Here we present data on freeze substituted samples of sporangia of the zygomycete Umbellopsis vinacea, basidia of Exobasidium camelliae var. gracilis, developing teliospores of the smut Sporisorium sorghi, germinating teliospores of the rust Gymnosporangium clavipes, germinating conidia of the deuteromycete Cercosporidium personatum, and developing ascospores of Ascodesmis nigricans.Spores of G. clavipes and C. personatum were deposited on moist pieces of sterile dialysis membrane where they hydrated and germinated. Asci of A. nigricans developed on pieces of dialysis membrane lying on nutrient agar plates. U. vinacea was cultured on small pieces of agar-coated wire. In the plant pathogens E. camelliae var. gracilis and S. sorghi, a razor blade was used to remove smal1 pieces of infected host issue. All samples were plunged directly into liquid propane and processed for study according to Hoch.l Samples on dialysis membrane were flat embedded. Serial thin sections were cut using a diamond knife, collected on slot grids, and allowed to dry down onto Formvar coated aluminum racks. Sections were post stained with uranyl acetate and lead citrate.

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Immunocytochemistry after fast freeze fixation-freeze substitution: Advantages and limits

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157759 ◽

1990 ◽

Vol 48 (3) ◽

pp. 42-43

Author(s):

Marie-Thérèse Nicolas

Keyword(s):

Osmium Tetroxide ◽

Freeze Substitution ◽

Acrylic Resin ◽

Expected Improvement ◽

List Type ◽

Chemical Fixation ◽

Freeze Fixation ◽

Liquid Chemical ◽

Luciferase Enzyme ◽

Lower Background

An alternative to aqueous chemical fixation consists in immobilizing physically the specimen by freezing it as fast as possible without using any cryoprotectant. This Fast Freeze Fixation (FFF) followed by Freeze Substitution (FS) avoids osmotic artefacts due to the slow penetration of liquid chemical fixative. Associated with Immuno-Gold labeling (IGS), FFF-FS allows a more precise localization of antigens.Using the bioluminescent bacteria Vibrio harveyi, a comparison of IGS with an antibody directed against its luciferase (enzyme of the luminescent reaction) has been done after liquid chemical fixation versus FFFFS. This later technique, beside an expected improvement of the ultrastructure always shows a better preservation of antigenicity and a lower background. In the case of FFF-FS technique (Figure 3):–labeling in acrylic resin (LRWhite) is 2 to 4 fold more intense than in epoxy resin (Epon),–but the ultrastructure is always better in Epon.–but the ultrastructure is always better in Epon.–The addition of fixatives in the substitution medium, results in a decrease of labeling which is more important in the case of a mixture of fixatives than with osmium tetroxide alone; with one exception: the substitution with glutaraldehyde which produces a dramatic increase in the density of the labeling but also, at the same time, a swelling of the cells of about 30%.

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Elemental analysis of human erythrocytes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015318x ◽

1989 ◽

Vol 47 ◽

pp. 242-243

Author(s):

S. A. Livesey ◽

A. A. del Campo ◽

E. S. Griffey ◽

D. Ohlmer ◽

T. Schifani ◽

...

Keyword(s):

Sample Preparation ◽

Elemental Analysis ◽

Human Erythrocyte ◽

Spectroscopic Analysis ◽

Model System ◽

Human Erythrocytes ◽

List Type ◽

Chemical Fixation ◽

Ethanol Dehydration ◽

Lowicryl K4m

The aim of this study is to compare methods of sample preparation for elemental analysis. The model system which is used is the human erythrocyte. Energy dispersive spectroscopic analysis has been previously reported for cryofixed and cryosectioned erythrocytes. Such work represents the reference point for this study. The use of plastic embedded samples for elemental analysis has also been documented. The work which is presented here is based on human erythrocytes which have been either chemically fixed and embedded or cryofixed and subsequently processed by a variety of techniques which culminated in plastic embedded samples.Heparinized and washed erythrocytes were prepared by the following methods for this study :(1). Chemical fixation in 4% paraformaldehyde/0.25% glutaraldehyde/0.2 M sucrose in 0.1 M Na cacodylate, pH 7.3 for 30 min, followed by ethanol dehydration, infiltration and embedding in Lowicryl K4M at -20° C.

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Intracellular structures of rapid frozen biological samples observed by high-resolution low-temperature Scanning Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155657 ◽

1989 ◽

Vol 47 ◽

pp. 736-737

Author(s):

T. Inoué ◽

H. Koike

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Low Temperature ◽

Liquid Nitrogen ◽

Specimen Preparation ◽

Chemical Fixation ◽

Brown Adipose ◽

Critical Point Drying ◽

Temperature Scanning ◽

Scanning Electron

Low temperature scanning electron microscopy (LTSEM) is useful to avoid artifacts such as deformation and extraction, because specimens are not subjected to chemical fixation, dehydration and critical-point drying. Since Echlin et al developed a LTSEM, many techniques and instruments have been reported for observing frozen materials. However, intracellular structures such as mitochondria and endoplasmic reticulum have been unobservable by the method because of the low resolving power and inadequate specimen preparation methods. Recently, we developed a low temperature SEM that attained high resolutions. In this study, we introduce highly magnified images obtained by the newly developed LTSEM, especially intracellular structures which have been rapidly frozen without chemical fixation.[Specimen preparations] Mouse pancreas and brown adipose tissues (BAT) were used as materials. After the tissues were removed and cut into small pieces, the specimen was placed on a cryo-tip and rapidly frozen in liquid propane using a rapid freezing apparatus (Eiko Engineering Co. Ltd., Japan). After the tips were mounted on the specimen stage of a precooled cryo-holder, the surface of the specimen was manually fractured by a razor blade in liquid nitrogen. The cryo-holder was then inserted into the specimen chamber of the SEM (ISI DS-130), and specimens were observed at the accelerating voltages of 5-8 kV. At first the surface was slightly covered with frost, but intracellular structures were gradually revealed as the frost began to sublimate. Gold was then coated on the specimen surface while tilting the holder at 45-90°. The holder was connected to a liquid nitrogen reservoir by means of a copper braid to maintain low temperature.

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Lectin-Induced RBC Agglutination: Chemical vs. Cryofixation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002920 ◽

1980 ◽

Vol 38 ◽

pp. 664-665

Author(s):

Dean A. Handley ◽

Lanping A. Sung ◽

Shu Chien

Keyword(s):

Lectin Binding ◽

Freeze Substitution ◽

Electrostatic Charge ◽

Geometric Parameters ◽

Comparative Approach ◽

Chemical Fixation ◽

Cell Surfaces ◽

Minimal Cell ◽

Membrane Stiffness ◽

Cell Contour

RBC agglutination by lectins represents an interactive balance between the attractive (bridging) force due to lectin binding on cell surfaces and disaggregating forces, such as membrane stiffness and electrostatic charge repulsion (1). During agglutination, critical geometric parameters of cell contour and intercellular distance reflect the magnitude of these interactive forces and the size of the bridging macromolecule (2). Valid ultrastructural measurements of these geometric parameters from agglutinated RBC's require preservation with minimal cell distortion. As chemical fixation may adversely influence RBC geometric properties (3), we used chemical fixation and cryofixation (rapid freezing followed by freeze-substitution) as a comparative approach to examine these parameters from RBC agglutinated with Ulex I lectin.

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