An Introduction to the Use of High Pressure Freezing/Freeze Substitution Fixation for TEM Studies of Biological Samples

2004 ◽  
Vol 10 (S02) ◽  
pp. 1268-1269
Author(s):  
Charles W. Mims ◽  
Elizabeth A. Richardson
Keyword(s):  
High Pressure ◽  
Biological Samples ◽  
Freeze Substitution ◽  
High Pressure Freezing

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

High-pressure freezing/freeze substitution of plant cells

1991 ◽  
Vol 49 ◽  
pp. 60-61
Author(s):  
J.Z. Kiss ◽  
L.A. Staehelin
Keyword(s):  
High Pressure ◽  
Biological Samples ◽  
Cell Structure ◽  
Plant Cells ◽  
Freeze Substitution ◽  
Specimen Preparation ◽  
High Pressure Freezing ◽  
New Information ◽  
Cold Metal ◽  
And Function

Electron microscopy of chemically fixed plant tissues has lead to important insights into the relationship between structure and function of plant cells. However, the slow rate of chemical fixation (seconds to minutes) potentially permits numerous artifacts to be induced. Most of these limitations ofs chemical fixatives can be overcome by the use of cryofixation techniques since cell structure is stabilized rapidly (milliseconds). Several types of cryofixation techniques have been developed such as cold metal block freezing and propane jet freezing. Although application of these techniques has yielded exciting new information, they are limiting in that specimens can be preserved only to a relatively shallow depth (approx. 40 μm). In contrast, under optimal conditions, high pressure freezing (HPF) at 2100 bar can produce excellent freezing of biological samples up to 600 μm in thickness. Since a commercial HPF apparatus has only recently become available, the number of systematic structural studies of biological samples utilizing HPF is still rather limited, and basic questions concerning specimen preparation and processing, HPF artifacts, and interpretation of images need to be addressed.

Low-temperature evaluation of high-pressure frozen biological samples by TEM cryosections and high-resolution SEM

1993 ◽  
Vol 51 ◽  
pp. 124-125
Author(s):  
Martin Müller
Keyword(s):  
High Pressure ◽  
Biological Samples ◽  
Structural Information ◽  
Freeze Substitution ◽  
Living System ◽  
Pressure Effects ◽  
High Yield ◽  
High Pressure Freezing ◽  
Direct Pressure ◽  
Cellular Water

The unique ability of electron microscopy in biology is its power to describe and integrate structural details down to molecular dimensions within the context of a complex living system.Structural information closely related to the living state may be achieved by cryoimmobilisation techniques that vitrify the cellular water and, at the same time rapidly arrest all physiological processes.High-pressure freezing (Müller and Moor 198, Moor 1987) is at present the only practical way of cryofixing larger non-pretreated samples up to a thickness of 500 μm. A very high yield of adequately frozen specimens was demonstrated in TEM using suspensions of microorganisms as well as plant and animal tissue (i. e. no detectable effects of ice crystal damage are visible after freeze substitution) (Studer et al. 1989). High-pressure freezing can vitrify biological samples sometimes up to a thickness of 200μm, as extrapolated from electron diffraction of cryosections (Michel et al 1991) The actual thickness that can be vitrified depends on the specimen, particularly on the presence of substances that exhibit cryoprotectant activity and also depends on the optimum transfer of pressure and cold to the sample and on the sample geometry (thickness and shape of the aquous layer, and the mass of the specimen planchettes necessery to protect the biological sample from direct pressure effects).

Jet freezing of cells and tissues with and without cryoprotectants

1993 ◽  
Vol 51 ◽  
pp. 106-107
Author(s):  
M.V. Parthasarathy ◽  
Carole Daugherty ◽  
T. Müller
Keyword(s):  
High Pressure ◽  
Biological Samples ◽  
Plant Cells ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
High Quality ◽  
The Past ◽  
Copper Specimen ◽  
Cell Structures

For the past several years cryofixation/freeze-substitution techniques have become valuable alternatives to chemical fixation of biological specimens. The superiority of cryofixation in preserving labile cell structures has been documented in several studies. Commercially available jet freezers and the BAL-TEC HPM010 high pressure freezer have extended high quality cryofixation from monolayer cells to cells relatively deep inside tissues. High pressure freezing can theoretically freeze biological materials of 0.5 mm thickness without the use of cryoprotectants and propane jet freezing is reported to freeze biological samples up to 40 μm in thickness without cryoprotection. Although high pressure freezing is the obvious method of choice for freezing large biological samples, its high cost combined with its apparent inability to consistently preserve microfilaments in some plant cells has prompted us to explore the capability of jet freezing to yield well frozen samples with and without cryoprotectants.We used the commercially available jet freezer JFD 030 (BAL-TEC) to obtain our results. Tightly pelleted cells sandwiched between 0.1 mm thick copper specimen carriers normally froze well without any cryoprotectants, after propane jet freezing (Figs. 1-2).

scholarly journals High Pressure Freezing; Benefits and Limitations

1998 ◽  
Vol 6 (9) ◽  
pp. 14-15
Author(s):  
Robert R. Wise
Keyword(s):  
High Pressure ◽  
Biological Activity ◽  
Liquid Nitrogen ◽  
Biological Samples ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
High Pressure Freezing ◽  
Dry Ice ◽  
Water Ice ◽  
Subsequent Processing

Rapid freezing of biological samples for subsequent processing and examination in the TEM has been around since the 1960's. Freezing in general, is advantageous because it can provide an instantaneous cessation of all biological activity and hall ultrastructural rearrangement The freezing ‘fixes” biological structures in water ice until such time that the ice can be substituted for a chemical fixative. Freezing is typically conducted at liquid nitrogen temperatures (-196°C), and freeze substitution (FS) is then carried out over days to weeks on dry ice (-80°C) (Severs el. al., 1995).

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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