High pressure freezing techniques for ultrastructural studies on fission yeast

Freeze substitution techniques can provide exceptional fixation of many types of small biological materials. Ultrastructural studies on the Foraminiferida (marine rhizopods) traditionally have used standard chemical fixation protocols. The relatively large size (-25 μm - several cm) and the presence of a mineralized shell in most taxa precludes the application of many cryo-techniques. High pressure freezing, however, provides a method for freezing organisms as large as many of the smaller foraminifera often without extensive ice damage. Gamonts (sexual generation) from three representatives of the suborder Astrorhizina ﹛Myxotheca sp., Cribrothalammina alba, and Hyperammina sp.) were selected because they are among the most primitive foraminifera and have only very finely agglutinated shells with non-mineralized, organic cements. Myxotheca sp. and C. alba were originally collected from salt marsh environments on Sapelo Island, Georgia, and Hyperammina sp. was isolated from fine-grained carbonate sediments of the Florida Keys.

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The Envelope of Intracellular African Swine Fever Virus Is Composed of a Single Lipid Bilayer

Journal of Virology ◽

10.1128/jvi.00194-08 ◽

2008 ◽

Vol 82 (16) ◽

pp. 7905-7912 ◽

Cited By ~ 20

Author(s):

Philippa C. Hawes ◽

Christopher L. Netherton ◽

Thomas E. Wileman ◽

Paul Monaghan

Keyword(s):

High Pressure ◽

Lipid Bilayer ◽

Lipid Membrane ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Viral Envelope ◽

Fever Virus ◽

Chemical Fixation ◽

High Pressure Freezing ◽

Ultrastructural Studies

ABSTRACTAfrican swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P< 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084624 ◽

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.

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Cryosectioning plant roots prepared by high-pressure freezing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127748 ◽

1987 ◽

Vol 45 ◽

pp. 662-663

Author(s):

R.E. Crang ◽

M. Mueller ◽

K. Zierold

Keyword(s):

High Pressure ◽

Cell Walls ◽

Plant Tissues ◽

Ice Crystal ◽

High Pressure Freezing ◽

Vitreous State ◽

Radial Depth ◽

Irregular Topography ◽

Considerable Depth ◽

Conventional Freezing

Obtaining frozen-hydrated sections of plant tissues for electron microscopy and microanalysis has been considered difficult, if not impossible, due primarily to the considerable depth of effective freezing in the tissues which would be required. The greatest depth of vitreous freezing is generally considered to be only 15-20 μm in animal specimens. Plant cells are often much larger in diameter and, if several cells are required to be intact, ice crystal damage can be expected to be so severe as to prevent successful cryoultramicrotomy. The very nature of cell walls, intercellular air spaces, irregular topography, and large vacuoles often make it impractical to use immersion, metal-mirror, or jet freezing techniques for botanical material.However, it has been proposed that high-pressure freezing (HPF) may offer an alternative to the more conventional freezing techniques, inasmuch as non-cryoprotected specimens may be frozen in a vitreous, or near-vitreous state, to a radial depth of at least 0.5 mm.

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High-pressure freezing and freeze substitution of plant tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124148 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161011 ◽

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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