Growth of ice crystals in frozen specimens

Nei, Tokio

doi:10.1111/j.1365-2818.1973.tb04675.x

Cited by 42 publications

(11 citation statements)

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“…In some cases, components of the cell may act as natural cryoprotectants. High recrystallization temperatures of 204 K and even 230 K are reported for frost-hardy plant cells (Sakai et al, 19681, yeast cells (Bank, 1973), human red blood cells (Nei, 1973) and rapidly frozen silk moth antennae (Steinbrecht, 1985a). These data are also compatible with successful electron microscopic studies on the ultrastructural preservation of biological cells and tissue prepared by freeze substitution around 190 K. Some aspects of fine structural results are discussed in the Applications section.…”

Section: Temperaturesupporting

confidence: 75%

Impact of freeze substitution on biological electron microscopy

Hippe-Sanwald

1993

Microscopy Res & Technique

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Considering the increasing necessity for improved preparation techniques in biological electron microscopy as a basis for the identification and localization of cellular substances within the compartments of the cell, this review is focussed on the method of freeze substitution as an important link between the cryofixation (ultrarapid freezing) and resin embedding of biological specimens. The theory and practice of freeze substitution is summarized with particular interest in the physical and thermodynamic as well as in the chemical basis of this technique. A survey of practical aspects of the technical process of freeze substitution concerning the equipment and various protocols successfully applied in biological systems is also given. The main advantage of freeze substitution versus conventional chemical fixation is seen in the maintenance of the hydration shell of molecules and macromolecular structures. This results in an improved fine structural preservation, superior retention of the antigenicity of proteins and decreased loss of unbound, diffusible cellular components. Examples of excellent visualization of the ultrastructure of macromolecular complexes (nucleic acids, extracellular material, membranes etc.), small organisms (bacteria, algae, cyanobacteria and fungi) and large biological samples such as plant and animal tissue as well as the plant-pathogen (fungus) interface and infection structures are presented. Recent data on the molecular characterization of freeze-substituted biological tissue are exemplified with special emphasis on the subcellular detection of soluble components (elements, lipids, proteins and drugs) and the inter-/intracellular localization of proteins including foreign proteins in transgenic plants. The molecular analysis of freeze-substituted specimens is achieved by the combination of low temperature preparation techniques in biological electron microscopy with various detection methods such as X-ray microanalysis, immunocytochemistry and high resolution autoradiography.

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Section: Temperaturesupporting

confidence: 75%

Impact of freeze substitution on biological electron microscopy

Hippe-Sanwald

1993

Microscopy Res & Technique

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“…However, the rate of loss of ice from a frozen biological specimen is slower than from pure ice, so a longer freeze‐drying time, or a higher temperature, may be required. Nei (1973) has shown that, in red cells, recrystallization of ice begins at temperatures above 193 K, so primary freeze‐drying needs to be complete before this temperature is reached. Our results show that for brass blocks of either weight there is sufficient time at the low temperatures for the primary stage of freeze‐drying to be completed.…”

Section: Discussionmentioning

confidence: 99%

Long freeze‐drying times are not necessary during the preparation of thin sections for X‐ray microanalysis

Warley

Skepper

2000

Journal of Microscopy

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SummaryThe temperature pro®le that occurs when a brass block warms up in a vacuum evaporation unit was determined. Freshly drawn human blood was concentrated by centrifugation, and the pellet was cryo®xed and cryosectioned. The cryosections were subject to different freeze-drying protocols, using a freeze-drier with a temperature-controlled stage, to determine the effect of freeze-drying time on element distribution. Spectra were collected by spot analyses at various distances across the interface between red cells and plasma. Concentrations of sodium were high and variable outside the cell and low in the cell interior, with potassium showing the reverse distribution. The number of counts under the iron peak closely followed the potassium distribution. The concentration of sodium was higher than expected at 40 nm inside the cell membrane. This was attributed to the formation of ice crystals at the interface between the cells and plasma during cryo®xation and the use of a wide probe size.

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“…Most volume of plant cell is occupied by central vacuole that contains freezable water. Cell damages during freezing and subsequent thawing can be caused, on the one hand, by the formation of intracellular ice crystals with acute facets disintegrating cell membranes [3,4], and, on the other hand, by dehydration. Therefore the most dangerous process, which may occur during freezing is intracellular ice formation.…”

Section: Methodical Basesmentioning

confidence: 99%