An Improved Method for Determining Ice Fabrics

AbstractIn ice-fabric studies, use of an accessory plate is necessary in conjunction with the Rigsby universal stage to discriminate between the a- and c-axes of an ice crystal when the orientation of the c-axis is parallel or nearly parallel to the stage. The techniques described permit orientation of the c-axis regardless of its orientation.

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The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance.

The Journal of Cell Biology ◽

10.1083/jcb.99.5.1655 ◽

1984 ◽

Vol 99 (5) ◽

pp. 1655-1668 ◽

Cited By ~ 88

Author(s):

P C Bridgman ◽

T S Reese

Keyword(s):

Cultured Cells ◽

Ground Substance ◽

Ice Crystal ◽

Improved Method ◽

Intact Cells ◽

Cultured Fibroblasts ◽

Cytoplasmic Proteins ◽

Granular Matrix ◽

Cytoplasmic Ground Substance ◽

Xenopus Laevis Embryos

Cultured fibroblasts or epithelial cells derived from Xenopus laevis embryos were directly frozen, freeze-substituted by an improved method, and then either critical-point-dried and viewed as whole mounts, or embedded and thin sectioned. In thin regions of these cells, where ice crystal artifacts are absent, the cytoplasm consisted of a dense, highly interconnected meshwork of filaments, embedded in a finely granular ground substance. The meshwork in directly frozen, intact cells was compared with that in cells that were lysed (physically, with detergents, or with filipin), or fixed with glutaraldehyde before freezing. Although filaments tended to be less numerous in lysed cells, their overall organization was the same as that in intact cells. However, fixation with glutaraldehyde before freezing distorted the meshwork to variable degrees depending on the osmolarity of the fixation buffer, and also obscured the granular ground substance which is obvious in directly frozen cells. With optimal preparative methods, the cytoplasm of these directly frozen cells is shown to consist of a cytoskeleton composed of discrete interwoven filaments interconnected by numerous finer filaments and a readily extractable granular matrix which presumably represents aggregations of cytoplasmic proteins.

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River-ice Fabrics: Preliminary Results

Journal of Glaciology ◽

10.3189/s0022143000013125 ◽

1971 ◽

Vol 10 (58) ◽

pp. 151-152

Author(s):

N.P. Lasca

Keyword(s):

Preliminary Data ◽

Stream Flow ◽

River Ice ◽

Ice Crystal ◽

Preliminary Results ◽

Ice Surface ◽

Crystal Orientations ◽

Response To Stress ◽

Ice Fabrics

AbstractPreliminary data indicate that fabric is developed in river ice. Crystal orientations are usually sub-parallel to the ice surface, and develop partly in response to stress generated by stream flow.

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River-ice Fabrics: Preliminary Results

Journal of Glaciology ◽

10.1017/s0022143000013125 ◽

1971 ◽

Vol 10 (58) ◽

pp. 151-152 ◽

Cited By ~ 4

Author(s):

N.P. Lasca

Keyword(s):

Preliminary Data ◽

Stream Flow ◽

River Ice ◽

Ice Crystal ◽

Preliminary Results ◽

Ice Surface ◽

Crystal Orientations ◽

Response To Stress ◽

Ice Fabrics

AbstractPreliminary data indicate that fabric is developed in river ice. Crystal orientations are usually sub-parallel to the ice surface, and develop partly in response to stress generated by stream flow.

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CRYSTAL ORIENTATION IN ICE SHEETS

Canadian Journal of Physics ◽

10.1139/p58-050 ◽

1958 ◽

Vol 36 (4) ◽

pp. 494-502 ◽

Cited By ~ 37

Author(s):

F. G. J. Perey ◽

E. R. Pounder

Keyword(s):

Surface Layer ◽

Crystal Size ◽

Pure Water ◽

Ice Sheets ◽

Gradual Change ◽

Ice Crystal ◽

Organic Additives ◽

One Dimensional ◽

The Mean ◽

Universal Stage

Crystal size and orientation in ice sheets frozen under one-dimensional cooling from melts of pure water or melts containing traces of organic additives were studied using a polariscope with a universal stage. The surface layer is found to consist of crystals with nearly vertical optic axes. Horizontal sections cut at various depths show a gradual change of the mean orientation of the optic axes towards the horizontal. The change to horizontal inclinations is almost completed at depths of 4 or 5 cm. under the freezing conditions used, and occurs more rapidly with the melts containing additives. With additives the ice sheet shows another feature also, a transition layer about 0.5 cm. below the surface. In this layer the crystals are much smaller than in the surface layer above them and the number of crystals extending through this layer is small.An explanation of the observations is offered, in terms of preferred growth of an ice crystal in planes perpendicular to the optic axis. This provides a mechanism permitting more rapid growth of crystals with inclined axes.

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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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The “KIMWIPE” Effect in Ultra-Thin Cryosections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119119 ◽

1985 ◽

Vol 43 ◽

pp. 458-459

Author(s):

I. Taylor ◽

P. Ingram ◽

J.R. Sommer

Keyword(s):

Skeletal Muscle ◽

Electrical Stimulation ◽

Muscle Fibers ◽

Ice Crystals ◽

Crystal Formation ◽

Ice Crystal ◽

Thin Sections ◽

Skeletal Muscle Fibers ◽

Freeze Dried ◽

Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

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An Improved Method for the Preparation and TEM Examination of Sharp Pointed Tips used in APFIM and STM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100134107 ◽

1990 ◽

Vol 48 (2) ◽

pp. 102-103

Author(s):

E.A. Fischione ◽

P.E. Fischione ◽

J.J. Haugh ◽

M.G. Burke

Keyword(s):

Atom Probe ◽

Preparation Process ◽

Improved Method ◽

Ion Milling ◽

Polishing Process ◽

Probe Field ◽

Scanning Tunnelling ◽

Stm Tips ◽

Tunnelling Microscopy ◽

Ion Microscopy

A common requirement for both Atom Probe Field-Ion Microscopy (APFIM) and Scanning Tunnelling Microscopy (STM) is a sharp pointed tip for use as either the specimen (APFIM) or the probe (STM). Traditionally, tips have been prepared by either chemical or electropolishing techniques. Recently, ion-milling has been successfully employed in the production of APFIM tips [1]. Conventional electropolishing techniques are applicable to a wide variety of metals, but generally require careful manual adjustments during the polishing process and may also be time-consuming. In order to reduce the time and effort involved in the preparation process, a compact, self-contained polishing unit has been developed. This system is based upon the conventional two-stage electropolishing technique in which the specimen/tip blank is first locally thinned or “necked”, and subsequently electropolished until separation occurs.[2,3] The result of this process is the production of two APFIM or STM tips. A mechanized polishing unit that provides these functions while automatically maintaining alignment has been designed and developed.

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