Vanhecke Dimitri, Graber Werner, Studer Daniel
Institute of Anatomy, University of Bern, 3000 Bern 9, Switzerland.
Methods Cell Biol. 2008;88:151-64. doi: 10.1016/S0091-679X(08)00409-3.
The objective of modern transmission electron microscopy (TEM) in life science is to observe biological structures in a state as close as possible to the living organism. TEM samples have to be thin and to be examined in vacuum; therefore only solid samples can be investigated. The most common and popular way to prepare samples for TEM is to subject them to chemical fixation, staining, dehydration, and embedding in a resin (all of these steps introduce considerable artifacts) before investigation. An alternative is to immobilize samples by cooling. High pressure freezing is so far the only approach to vitrify (water solidification without ice crystal formation) bulk biological samples of about 200 micrometer thick. This method leads to an improved ultrastructural preservation. After high pressure freezing, samples have to be subjected to follow-up procedure, such as freeze-substitution and embedding. The samples can also be sectioned into frozen hydrated sections and analyzed in a cryo-TEM. Also for immunocytochemistry, high pressure freezing is a good and practicable way.
现代生命科学中的透射电子显微镜（TEM）的目标是在尽可能接近生物体的状态下观察生物结构。TEM样品必须很薄且要在真空中进行检测；因此只能研究固体样品。制备TEM样品最常见且流行的方法是在检测前对其进行化学固定、染色、脱水并嵌入树脂中（所有这些步骤都会引入相当多的假象）。另一种方法是通过冷却固定样品。到目前为止，高压冷冻是使约200微米厚的大量生物样品玻璃化（水固化而不形成冰晶）的唯一方法。这种方法能改善超微结构的保存。高压冷冻后，样品必须经过后续处理，如冷冻置换和包埋。样品也可以切成冷冻水合切片并在低温TEM中进行分析。同样，对于免疫细胞化学来说，高压冷冻也是一种良好且可行的方法。
