Cryofixation Without Pretreatment at Ambient Pressure
Cryofixation Without Pretreatment at Ambient Pressure
复制标题
在环境压力下无需预处理的冷冻固定
DOI：
--
复制
发表时间：
1987
期刊：
影响因子：
--
通讯作者：
K. Neumann
中科院分区：
文献类型：
--
作者：
H. Sitte;L. Edelmann;K. Neumann
几乎在世纪前，生物或医学对象在冷冻状态下使用，以加速病理诊断（立即切片）并更好地保持其化学组成（Altmann 1890）。这种替代化学固定的方法在本世纪上半叶变得越来越重要，这是因为可以使用液化空气作为冷却剂（“冷冻剂”），以及已经开发出了有效的低温恒温器冷却系统（参见Gersh 1932; Simpson 1941; Eranko 1954; Kulenkampff 1955; Neumann 1958）。尝试冷冻潮湿的物体进行电子显微镜检查显示，在正常的大气压和最有利的条件下，只有大约一个。30-μm的边界区可以完全冻结（参见Sitte 1979; Plattner and Bachmann 1982; Robards and Sleytr 1985）。在更大的深度，混合的等离子体相分离。这些隔离室的大小形成的增长冰晶内的标本增加得如此之快，更深的层不能用于电子显微镜。保存完好的边界区的深度可以在不进行化学预处理的情况下通过施加约2100巴的高压而增加到最多300 μm（Muller和摩尔，1984年）。通过使用防冻剂（“冷冻保护剂”），这些限度可以大大扩展：甘油已被证明是冷冻断裂/冷冻蚀刻法的有效防冻剂（摩尔和Muhlethaler 1963），蔗糖用于冷冻切片术（参见Bernhard和Leduc 1967; Tokuyasu 1973; Griffiths等人1984）。
Almost a century ago biological or medical objects were used in their frozen state in order to accelerate pathological diagnosis (immediate section) and to better maintain their chemical constitution (Altmann 1890). This alternative to chemical fixation gained in importance during the first half of this century due to the availability of liquefied air used as coolant (“cryogen”) as well as to the fact that effective cooling systems for cryostats had been developed (see e.g. Gersh 1932; Simpson 1941; Eranko 1954; Kulenkampff 1955; Neumann 1958). Attempts to freeze wet objects for electron microscopy revealed that at normal atmospheric pressure and under the most favourable conditions only an approx. 30- μm border zone can be perfectly frozen (see e.g. Sitte 1979; Plattner and Bachmann 1982; Robards and Sleytr 1985). At greater depths the mixed plasmatic phases segregate. The size of these segregation compartments formed by the growing ice crystals within the specimen increases so rapidly that deeper layers cannot be used for electron microscopy. The depth of the well-preserved border zone can be increased without chemical pretreatment to, at the most, 300 μm by applying high pressures of about 2100 bar (Muller and Moor 1984). These limits can be considerably extended by the use of anti-freezing agents (“cryoprotectants”): glycerol has proved to be an effective anti-freeze for the freeze-fracture/freeze-etch method (Moor and Muhlethaler 1963) and saccharose in cryoultramicrotomy (see e.g. Bernhard and Leduc 1967; Tokuyasu 1973; Griffiths et al. 1984).