Frederik P M, Stuart M C, Bomans P H, Busing W M
Department of Pathology, University of Limburg, Maastricht, The Netherlands.
J Microsc. 1989 Jan;153(Pt 1):81-92. doi: 10.1111/j.1365-2818.1989.tb01469.x.
Thin films of surface-active compounds, with or without particulate material, can be obtained by immersing and withdrawing a bare specimen grid from a solution/suspension of the compound. Immediately after withdrawing the grid, thinning of the film starts. Thinning is initially powered by gravity and capillary forces and will proceed in thin films (less than 100 nm) driven by intermolecular forces until the London-van der Waals attractive forces come to an equilibrium with electrostatic repulsion of similarly charged surfaces of the film. With small unilamellar vesicles prepared from the phospholipid dimyristoyl phosphatidyl choline (DMPC) the draining behaviour of these films was studied by cryo-electron microscopy. Small unilamellar vesicles were observed within the film as well as the coalescence of these vesicles into sheets ('leaky' membrane fusion). Sheets dominate the images when films are allowed to drain for longer periods (greater than 3 min). Thin films were formed on grids from catalase crystals suspended in a DMPC suspension and vitrified by cooling. High-resolution information was obtained by electron diffraction at low temperature and under low-dose conditions from catalase crystals surrounded by small vesicles as well as from catalase crystals surrounded by sheets of DMPC. In the latter case the water content drops from 99% (DMPC in small vesicles) to less than 30% (DMPC in sheets) during draining. Ferritin was added to a DMPC suspension and thin films were prepared and vitrified. After prolonged draining ferritin molecules were deposited in layers with a stepwise increase in thickness. Draining of thin films has thus a dehydrating effect as well as a sorting and ordering effect. These effects must be considered when using surface-active compounds at air-water interfaces as a slide and cover slip for electron microscopy.
通过将裸露的标本网格浸入并取出表面活性化合物的溶液/悬浮液中，可以获得含有或不含有颗粒物质的表面活性化合物薄膜。取出网格后，薄膜立即开始变薄。变薄最初由重力和毛细管力驱动，在由分子间力驱动的薄膜（小于100纳米）中继续进行，直到伦敦-范德华吸引力与薄膜相似带电表面的静电排斥力达到平衡。用磷脂二肉豆蔻酰磷脂酰胆碱（DMPC）制备的小单层囊泡，通过冷冻电子显微镜研究了这些薄膜的排水行为。在薄膜中观察到了小单层囊泡，以及这些囊泡聚合成片（“渗漏”膜融合）。当薄膜排水较长时间（大于3分钟）时，片层在图像中占主导地位。薄膜由悬浮在DMPC悬浮液中的过氧化氢酶晶体在网格上形成，并通过冷却进行玻璃化处理。通过低温和低剂量条件下的电子衍射，从被小囊泡包围的过氧化氢酶晶体以及被DMPC片层包围的过氧化氢酶晶体中获得了高分辨率信息。在后一种情况下，排水过程中水含量从99%（小囊泡中的DMPC）降至小于30%（片层中的DMPC）。将铁蛋白添加到DMPC悬浮液中，制备并玻璃化薄膜。长时间排水后，铁蛋白分子分层沉积，厚度逐步增加。因此，薄膜的排水具有脱水作用以及分选和排序作用。在使用空气-水界面的表面活性化合物作为电子显微镜的载玻片和盖玻片时，必须考虑这些影响。
