冰冻切片的注意事项
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关于冷冻理论原文剪贴
原文作者：Mark Donovan and Henry Preston
Theory of freezing
The reasons for freezing a tissue sample are to provide a hardened matrix for sectioning or to preserve the morphological, biochemical or immunological properties of a cell or tissue. The use of a low temperature can eliminate many of the problems associated with standard practices of chemical fixation and paraffin or resin embedding. In practice however, the process of transforming water into ice can dramatically alter the physical and chemical structure of cells and tissue.
When pure water is cooled, large hexagonal ice crystals form as a result of homogeneous and heterogeneous nucleation and subsequent growth of ice crystal nuclei. The transformation of water into ice crystals occurs at a temperature of O°C and a pressure of 1 atmosphere. Where a very high rate of cooling exists, cubic rather than hexagonal ice crystals will form. These are far smaller and produce less distortion on formation. If the rate of cooling is increased further to within the order of 104K/sec small volumes of water can be solidified without the formation of ice crystals at all.
1 This solid form of ice, known as vitreous ice, exists in a temperature dependent, irreversible phase transition with cubic ice, hexagonal ice and water.
2 The critical temperatures at which these phase transitions occur have only been accurately determined for pure water3 but the values for intracellular water are considered to be significantly higher (fig 1).4-5
(fig 1).4-5：http://home.primus.com.au/royellis/fig1.html
The formation of large, hexagonal ice crystals as the result of slow freezing occurs firstly in the less concentrated extracellular fluid. This produces an osmotic difference between the extracellular and intracellular fluid which results in a loss of intracellular water and the subsequent shrinkage of the cell as the osmotic balance returns. The increased ionic concentration within the cell ruptures membranes and denatures the protoplasm. The eventual formation of ice crystals within the cell, when residual intracellular fluid freezes, may then mechanically fracture the cell. These effects are collectively known as ice crystal artefacts.
The damage produced by ice crystals depends upon the size and type of crystal formed during the freezing process. Large hexagonal ice crystals will produce major structural damage to cells and tissue whilst smaller cubic ice crystals cause less cellular damage. Ideally an extremely rapid cooling rate should be used as this will produce vitreous ice without crystal damage. The aim when freezing the sample, therefore, is to limit ice crystal formation as much as possible through control of the cooling rate.
Factors which effect the cooling rate of the sample include:
the absolute temperature of the cryogen.
the heat capacity of the cryogen.
extent of contact between cryogen and sample.
degree of heat exchange between cryogen and sample.
diffusion of heat through the sample.
Freezing of the specimen will require selection of an appropriate cryogen and procedures which maximise the heat exchange between cryogen and specimen. The cooling rate attainable is particularly influenced by specimen size. Above a critical specimen size optimal freezing will only occur to a certain depth and the cooling rate in the deeper parts of the sample will be slow enough to allow the formation of hexagonal ice crystals with subsequent tissue damage. It is possible to reduce the level of ice crystal formation through the use of cryoprotectants which reduce the rate of ice crystal nucleation through freezing point depression.
