High-pressure freeze fixation reveals novel features during ontogenesis of the vegetative cell in Ledebouria pollen: an ultrastructural and cytochemical study

1995 ◽  
Vol 73 (1-2) ◽  
pp. 1-10 ◽  
Author(s):  
Michael W. Hess
Keyword(s):  
High Pressure ◽  
Vegetative Cell ◽  
Spatial Organization ◽  
Spatial Relationship ◽  
Freeze Substitution ◽  
Structural Features ◽  
Protein Bodies ◽  
Lipid Digestion ◽  
Cytochemical Study ◽  
Freeze Fixation

The ultrastructure of the vegetative cell in the pollen of Ledebouria socialis Roth (Hyacinthaceae) was investigated from microspore mitosis to anthesis. As a result of the good preservation quality achieved with high-pressure freeze fixation and freeze substitution, novel structural features were observed. Extensive endomembrane compartments emerging at the onset of lipid and starch mobilization, were identified as protein bodies by using video-enhanced contrast light microscopy. Thus, proteins, apart from starch and lipids, represent a third class of important intermediary storage substances in developing pollen. The close spatial relationship between protein bodies, endoplasmic reticulum (ER), and storage lipids suggest that protein bodies and ER contribute to lipid digestion. Immediately prior to anthesis the protein bodies become transformed into unspecialized vacuoles as a result of the gradual dissolution of their contents; the formation of the protein bodies remains still to be elucidated. The ER proliferates extensively during pollen ontogenesis, thereby changing its ultrastructure and spatial organization. Microfilaments were detected during all developmental stages, in particular microtubule-associated single microfilaments. The microfilaments are likely to be composed of actin as shown by immunogold labeling.Key words: angiosperm pollen, freeze substitution, protein bodies, microfilaments, Hyacinthaceae.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

High-pressure freezing and freeze substitution of plant tissue

1992 ◽  
Vol 50 (1) ◽  
pp. 748-749
Author(s):  
William P. Sharp ◽  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Cell Structure ◽  
Leaf Tissue ◽  
Freeze Substitution ◽  
Crystal Formation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Components ◽  
Cold Metal ◽  
Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

High-pressure freezing and freeze substitution of teliospores of the rust fungus Gymnosporangium clavipes

1990 ◽  
Vol 48 (3) ◽  
pp. 692-693
Author(s):  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Room Temperature ◽  
Pathogenic Fungus ◽  
Rust Fungus ◽  
Freeze Substitution ◽  
Ice Crystals ◽  
High Pressure Freezing ◽  
Thin Walled Structures ◽  
Cytological Changes ◽  
Dextran Solution

The use of cryo-techniques for the preparation of biological specimens in electron microscopy has led to superior preservation of ultrastructural detail. Although these techniques have obvious advantages, a critical limitation is that only 10-40 μm thick cells and tissue layers can be frozen without the formation of distorting ice crystals. However, thicker samples (600 μm) may be frozen well by rapid freezing under high-pressure (2,100 bar). To date, most work using cryo-techniques on fungi have been confined to examining small, thin-walled structures. High-pressure freezing and freeze substitution are used here to analysis pre-germination stages of specialized, sexual spores (teliospores) of the plant pathogenic fungus Gymnosporangium clavipes C & P.Dormant teliospores were incubated in drops of water at room temperature (25°C) to break dormancy and stimulate germination. Spores were collected at approximately 30 min intervals after hydration so that early cytological changes associated with spore germination could be monitored. Prior to high-pressure freezing, the samples were incubated for 5-10 min in a 20% dextran solution for added cryoprotection during freezing. Forty to 50 spores were placed in specimen cups and holders and immediately frozen at high pressure using the Balzers HPM 010 apparatus.

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

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%.

scholarly journals Immunolocalization of type III collagen in human articular cartilage prepared by high-pressure cryofixation, freeze-substitution, and low-temperature embedding.

1995 ◽  
Vol 43 (4) ◽  
pp. 421-427 ◽  
Author(s):  
R D Young ◽  
P A Lawrence ◽  
V C Duance ◽  
T Aigner ◽  
P Monaghan
Keyword(s):  
High Pressure ◽  
Articular Cartilage ◽  
Polyclonal Antibodies ◽  
Freeze Substitution ◽  
Immunogold Electron Microscopy ◽  
Type Iii Collagen ◽  
Human Articular Cartilage ◽  
Chemical Fixation ◽  
Osteoarthritic Cartilage ◽  
Type Iii

We localized Type III collagen by immunogold electron microscopy in resin sections of intact normal and osteoarthritic human articular cartilage. Comparisons of antibody staining between tissue prepared by high-pressure cryofixation and freeze-substitution without fixatives and that exposed to conventional mild chemical fixation with paraformaldehyde showed that dedicated cryotechniques yielded superior preservation of epitopes that are modified by chemical fixation, and simultaneously provided good ultrastructural preservation. Type III collagen was detected with two polyclonal antibodies, one against the triple-helical domain of the molecule and a second against the more antigenic, globular amino pro-peptide domain, which in this collagen is retained in the extracellular matrix after secretion. Positive labeling was seen in association with the major interstitial fibrils, suggesting co-polymerization of Types III and II collagen in cartilage. Type III collagen could not be detected in aldehyde-fixed normal cartilage. In fixed osteoarthritic cartilage, Type III was detectable only when the antibody to the amino pro-peptide was employed. In contrast, high-pressure cryofixation and freeze-substitution preserved epitopes for both antibodies, permitting immunodetection of Type III collagen in normal and osteoarthritic cartilage. Cryotechniques offer exciting possibilities for significantly improving the immunolocalization of collagens and other fixative-sensitive antigens in situ.

scholarly journals Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution

2022 ◽  
Author(s):  
Martin Schauflinger ◽  
Tim Bergner ◽  
Gregor Neusser ◽  
Christine Kranz ◽  
Clarissa Read
Keyword(s):  
High Pressure ◽  
Potassium Permanganate ◽  
Lipid Membranes ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Block Face ◽  
Critical Aspects

AbstractHigh-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

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