Glaeser R M
Department of Molecular and Cell Biology, Stanley/Donner ASU, University of California, Berkeley 94720.
Ultramicroscopy. 1992 Oct;46(1-4):33-43. doi: 10.1016/0304-3991(92)90006-6.
The extreme degree of specimen flatness (i.e. planarity) required for high-resolution electron diffraction and electron microscopy at high tilt angles cannot be realized with thin, sheet-like crystals of biological macromolecules, just on the basis of the intrinsic stiffness of the specimen itself. In an effort to improve the rate of success at which suitably flat specimens are prepared, this paper analyzes several different factors that can either limit or enhance the specimen flatness. If specimens are adsorbed (by attractive forces) to a support film, such as evaporated carbon, which itself is not flat to atomic dimensions, quantitative calculations show that it is quite likely that the specimen will be too wrinkled to be used for high-resolution studies. Adsorption to an air-water interface is more likely to result in the necessary degree of flatness. Repulsive interactions, which might be used to "sandwich" a specimen between two interfaces, are estimated to be too "soft", i.e. too long-range in character, to be effective. Finally, if only one edge of a specimen sticks firmly to a substrate, then surface tension forces can pull the specimen taut over the surface of the substrate, so that the specimen itself can be more flat than the surface of the substrate upon which it is deposited. A second, important consideration in many studies is the fact that cooling the specimen to low temperature can result in specimen wrinkling, because of the fact that the biological crystal has a much larger coefficient of thermal expansion than that of the evaporated carbon film. In this case one expects that cooling-induced wrinkling might be reduced by using a metal support grid which has a smaller thermal coefficient than that of the carbon film. The validity of this qualitative idea is supported by experiments which show that cooling-induced wrinkling of glucose-embedded purple membrane can be prevented if molybdenum grids are used rather than copper.
对于高分辨率电子衍射和高倾斜角电子显微镜而言，仅基于生物大分子薄片状晶体自身的固有刚度，无法实现所需的极高程度的样品平整度（即平面度）。为了提高制备出合适平整度样品的成功率，本文分析了几个可能限制或增强样品平整度的不同因素。如果样品通过吸引力吸附到支撑膜上，比如蒸发碳膜，而该支撑膜本身在原子尺度上并不平整，定量计算表明样品很可能会过于褶皱而无法用于高分辨率研究。吸附到气 - 水界面更有可能产生所需的平整度。据估计，可能用于将样品“夹”在两个界面之间的排斥相互作用过于“柔和”，即作用范围太长，因而无效。最后，如果样品只有一条边牢固地附着在基底上，那么表面张力可以将样品拉紧在基底表面，这样样品本身可能比其沉积的基底表面更平整。在许多研究中，另一个重要的考虑因素是，将样品冷却到低温会导致样品起皱，因为生物晶体的热膨胀系数比蒸发碳膜的热膨胀系数大得多。在这种情况下，可以预期通过使用热系数比碳膜小的金属支撑网格来减少冷却引起的起皱。实验支持了这一定性观点的有效性，实验表明，如果使用钼网格而非铜网格，就可以防止嵌入葡萄糖的紫膜因冷却而起皱。
