Visualization of the Cytoskeleton by Cryofixation, Immunoelectron Microscopy and 3-D Reconstruction Techniques

1990 ◽  
Vol 48 (3) ◽  
pp. 196-197
Author(s):  
Kent McDonald
Keyword(s):  
High Pressure ◽  
High Voltage ◽  
Freeze Substitution ◽  
Serial Sections ◽  
High Pressure Freezing ◽  
Area Of Interest ◽  
Frozen Embryos ◽  
Conventional Microscopy ◽  
Mutant Phenotypes ◽  
And Function

We are using various combinations of the following techniques to study cytoskeleton structure and function: cryofixation by high pressure freezing, double jet or plunge freezing; freeze substitution; cryosectioning of frozen hydrated or frozen substituted cells; EM immunolocalization; high voltage and conventional cryoelectron microscopy; serial sectioning (thick and/or thin); and computer-mediated 3-D reconstructions from serial sections.One area where many of these tools and techniques are called into play is in the ultrastructural analysis of developmental mutant phenotypes in Drosophila. Staged embryos are ultrarapidly frozen by high pressure freezing then divided into three groups for further processing. One group is freeze-substituted in osmium-acetone then flat embedded and sectioned for examination by high voltage or conventional microscopy. This allows us to evaluate the quality of cryofixation and the percentage of well frozen embryos. From this material we will also cut serial sections which will be used to make 3-D models of the area of interest. The second group of frozen embryos will be put directly into a Reichert FC4 cryoultramicrotome and sectioned at -150 C. The cryosections are then examined in the cryostage of the high voltage EM. The third group is freeze-substituted in methanol plus aldehyde fixatives in preparation for immunological studies.

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.

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.

Ultrastructure of the Infection of Poinsettia by Oidium SP. using High Pressure Freezing and Freeze Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 690-691
Author(s):  
G. J. Celio ◽  
E. A. Richardson ◽  
C. W. Mims
Keyword(s):  
High Pressure ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Euphorbia Pulcherrima ◽  
High Pressure Freezing ◽  
Thin Sections ◽  
Transmission Electron ◽  
Dextran Solution ◽  
Leaf Disks ◽  
Diamond Knife

Cryofixation is becoming more widely used to study host-pathogen relationships in fungal diseases of plants. This presentation describes results we have obtained using high pressure freezing and freeze substitution to study powdery mildew disease of poinsettia ﹛Euphorbia pulcherrima) caused by Oidium sp.Approximately 0.5 mm leaf disks bearing sporulating colonies of Oidium sp. were excised and placed in a 15% dextran solution contained in brass planchets. Samples were frozen using a Balzer's HPM 010 High Pressure Freezing Machine and substituted according to the procedures of Hoch.6 Thin sections of embedded leaves were cut using a diamond knife, collected on gold slot grids, and placed on formvar-coated racks. Sections were poststained with uranyl acetate and lead citrate and examined using a Zeiss EM 902A transmission electron microscope.Outstanding preservation of haustoria, the specialized nutrient-absorbing structures produced in host epidermal cells by Oidium, was obtained. Both young, unlobed (Fig. 1) as well as mature, highly lobed (Fig. 2) haustoria were observed.

Cryo-SEM and TEM of High Pressure Frozen Cells - Some Technical Contributions

2001 ◽  
Vol 7 (S2) ◽  
pp. 728-729
Author(s):  
Paul Walther
Keyword(s):  
High Pressure ◽  
Physiological State ◽  
Electron Microscopic Observation ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Electron Microscopic ◽  
Biological Specimen ◽  
Freeze Fracturing

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.The figures show high pressure frozen bakers yeast saccharomyces cerevisiae in the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

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