scholarly journals Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure - preservation of proteoglycans in their native state.

1984 ◽  
Vol 98 (1) ◽  
pp. 277-282 ◽  
Author(s):  
E B Hunziker ◽  
R K Schenk
Keyword(s):  
High Pressure ◽  
Low Temperature ◽  
Freeze Substitution ◽  
Epiphyseal Cartilage ◽  
Cartilage Tissue ◽  
High Pressure Freezing ◽  
Intercellular Matrix ◽  
The Matrix ◽  
Protein Components ◽  
Ultrastructure Preservation

The extracellular matrix of epiphyseal cartilage tissue was preserved in a state believed to resemble closely that of native tissue following processing by high pressure freezing, freeze substitution, and low temperature embedding (HPF/FS). Proteoglycans (PG) were preserved in an extended state and were apparent as a reticulum of fine filamentous threads throughout the matrix. Within this network, two morphologically discrete components were discernible and identified with the carbohydrate and protein components of PG molecules. Numerous points of contact were clearly visible between components of the PG network and cross-sectioned collagen fibrils and also between PG components and chondrocytic plasmalemmata. These observations provide direct morphological indication that such relationships may exist in native epiphyseal cartilage tissue.

scholarly journals Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure--implications for the theories of mineralization and vascular invasion.

1984 ◽  
Vol 98 (1) ◽  
pp. 267-276 ◽  
Author(s):  
E B Hunziker ◽  
W Herrmann ◽  
R K Schenk ◽  
M Mueller ◽  
H Moor
Keyword(s):  
High Pressure ◽  
Low Temperature ◽  
Vascular Invasion ◽  
Freeze Substitution ◽  
Electron Microscopic Examination ◽  
Cartilage Tissue ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
Electron Microscopic ◽  
Cell Destruction

Electron microscopic examination of epiphyseal cartilage tissue processed by high pressure freezing, freeze substitution, and low temperature embedding revealed a substantial improvement in the preservation quality of intracellular organelles by comparison with the results obtained under conventional chemical fixation conditions. Furthermore, all cells throughout the epiphyseal plate, including the terminal chondrocyte adjacent to the region of vascular invasion, were found to be structurally integral. A zone of degenerating cells consistently observed in cartilage tissue processed under conventional chemical fixation conditions was not apparent. Hence, it would appear that cell destruction in this region occurs during chemical processing and is not a feature of cartilage tissue in the native state. Since these cells are situated in a region where tissue calcification is taking place, the implication is that the onset and progression of cartilage calcification are, at least partially, controlled by the chondrocytes themselves. The observation that the terminal cell adjacent to the zone of vascular invasion is viable has important implications in relation to the theory of vascular invasion. This may now require reconceptualization to accommodate the possibility that active cell destruction may be a precondition for vascular invasion.

scholarly journals In situ localization of cartilage extracellular matrix components by immunoelectron microscopy after cryotechnical tissue processing.

1987 ◽  
Vol 35 (6) ◽  
pp. 647-655 ◽  
Author(s):  
E B Hunziker ◽  
W Herrmann
Keyword(s):  
High Pressure ◽  
Low Temperature ◽  
Immunoelectron Microscopy ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
Morphological Examination ◽  
Extracellular Matrix Components ◽  
Matrix Components

Localization and distribution of proteoglycans within rat growth plate cartilage were investigated by immunoelectron microscopy. By use of a mixture of three monoclonal antibodies directed against chondroitin sulfate chains and of post-embedding staining by protein A-gold, the immunosensitivity and resolution achieved by electron microscopy within tissue processed by high-pressure freezing, freeze-substitution, and low-temperature embedding were compared with those in tissue preserved by three alternative procedures (i.e., mild chemical fixation in combination with either low-temperature embedding or conventional embedding, and high-pressure freezing and freeze-substitution followed by conventional embedding). The loss of matrix components incurred during each stage of high-pressure freezing, freeze-substitution, and low temperature embedding was also determined by measuring the loss of [35S]-proteoglycans from tissue labeled in vivo, and the results compared with previously determined estimates for tissue processed using conventional techniques. Immunosensitivity, determined as the number of gold particles per unit area, was highest in tissue processed by high-pressure freezing, freeze substitution, and low-temperature embedding. Comparable results (with a reduction of only 3-7%) were achieved within tissue preserved by mild chemical fixation followed by low-temperature embedding. In both procedures where conventional embedding was adopted, sensitivity was considerably reduced (by 51% for high-pressure freezing and freeze substitution and by 74% for mild chemical fixation). Loss of matrix components was negligible during all stages of high-pressure freezing, freeze-substitution, and low-temperature embedding. Such information, and that derived from morphological inspection of the various matrix compartments in cartilage processed by high-pressure freezing, freeze-substitution, and low-temperature embedding (J Cell Biol 98:277, 1984), together demonstrate that application of this technique results in successful immobilization of proteoglycans in situ within cartilage matrix. Although loss of proteoglycans from mildly fixed cartilage embedded under low-temperature conditions is minor, morphological examination of this tissue reveals considerable shifting of proteoglycans within matrix compartments. Hence, even though immunosensitivity may be high, resolution is poor. The beauty of the high-pressure freezing, freeze-substitution, and low-temperature embedding technique is that it combines high immunosensitivity with precise localization of matrix components at the molecular level.

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.

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