Glaeser R M, Zilker A, Radermacher M, Gaub H E, Hartmann T, Baumeister W
Department of Molecular and Cell Biology, Donner Laboratory, University of California, Berkeley 94720.
J Microsc. 1991 Jan;161(Pt 1):21-45. doi: 10.1111/j.1365-2818.1991.tb03071.x.
It is generally agreed that surface-tension forces and the direct interaction between the specimen and either the air-water interface or the water-substrate interface can influence significantly the preparation of biological materials for electron microscopy. Even so, there is relatively little systematic information available that would make it possible to control surface-tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1 degree, and perhaps less than 0.2 degrees, over areas as large as 1 micron2 or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose-embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air-water interface rather than adsorption of the specimen to the substrate. Surface-tension forces at the air-water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air-water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.
人们普遍认为，表面张力以及标本与气-水界面或水-底物界面之间的直接相互作用会对用于电子显微镜观察的生物材料的制备产生显著影响。即便如此，可用于以定量方式控制表面张力和界面能的系统性信息相对较少。开展本研究的主要目的是更好地了解影响生物大分子大单层晶体标本平整度的因素。然而，我们研究中获得的数据总体上应有助于理解生物大分子标本的制备。通过电子衍射和高分辨率、高倾斜角电子显微镜进行数据收集，需要生物大分子薄晶体在至少1度、甚至可能小于0.2度的范围内保持平整，覆盖面积达1平方微米或更大。除了通过电子衍射实验凭经验确定能否在各种改性或未改性的碳支撑膜上制备出足够平整的标本外，我们还开始使用其他技术来表征相关表面以及标本与这些表面的相互作用。在将细菌视紫红质大单层晶体作为葡萄糖包埋标本制备在疏水碳膜上的具体案例中，得出的结论是，初始界面相互作用涉及标本吸附到气-水界面，而非标本吸附到底物。在这种情况下，气-水界面的表面张力以及标本与疏水碳之间明显的排斥相互作用似乎是影响标本平整度的主要因素。在更一般的情况下，在寻找理想的标本制备条件时，与底物或气-水界面的界面相互作用似乎可以通过多种方式进行调控。
