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.

Cryofixation of various biological samples by high pressure freezing

1992 ◽  
Vol 50 (1) ◽  
pp. 752-753
Author(s):  
LUCY RU-SIU YIN
Keyword(s):  
High Pressure ◽  
Biological Samples ◽  
High Rate ◽  
Crystal Formation ◽  
Chemical Fixation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Native State ◽  
Biological Specimen ◽  
Ice Crystal Formation

The ultimate aim of ultrastructural fixation of biological specimen is to preserve all the compartments in their native state. Cryofixation is a superior method than conventional chemical fixation in reaching this goal. However, ice crystal formation during cryofixation often damages the structures. High pressure (2100 bar) freezing provides a way to alter freezing properties while cool down the specimen at a relatively high rate, minimizing the ice crystal formation. Nearly vitrified samples(up to 500 um) have been obtained with this method. Samples in suspension tend to get lost during high pressure freezing. The low percentage (∼30%) of successfully cryofixed specimens can be improved if the sample completely fills the cavity of the metal specimen carriers in which the specimen is frozen. Various methods to overcome sample loss are reported in this study.

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.

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.

Electron microscopic observation of photoreceptor cells in directly inserted anesthetized Drosophila into a high‐pressure freezing unit

2018 ◽  
Vol 82 (3) ◽  
pp. 244-249
Author(s):  
Hitomi Terada ◽  
Yurika Saitoh ◽  
Kiyokazu Kametani ◽  
Masahiko Sakaguchi ◽  
Takeharu Sakamoto ◽  
...  
Keyword(s):  
High Pressure ◽  
Microscopic Observation ◽  
Electron Microscopic Observation ◽  
Photoreceptor Cells ◽  
High Pressure Freezing ◽  
Electron Microscopic
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