应用电子显微学方法研究蛋白质薄晶体结构,通常使用负染色方法。然而它存在缺陷:负染色剂在电子束照射下会发生变化,如醋酸铀体积减少达15％,这会引起蛋白质表面移动(P.Un-win)。其次由于负染色液与氨基酸残余侧链基因的化学作用。会出现正的或负的染色,使得图象难以解释(W.Chin)。最后负染色的样品大约只能获得15～20A的分辨率。如不进行染色,也有问题:象衬度非常弱;存在真空损伤——蛋白质从含水状态到电镜真空中,因干燥而引起损伤;最后是幅射损伤——蛋白质晶体对电子束非常敏感,典型的蛋白质晶体,大约在le/A~2的电子剂量时就开始被破坏(Stenn)。这样的剂量是远小于通常观察时的剂量。为解决这些问题,A.Klug等人采用下列方法:用相位衬度成象;用葡萄糖取代水介质,由于葡萄糖不挥发,有利于真空保 Electron microscopy methods are used to study protein thin crystal structures, usually using negative staining methods. However, it has the disadvantage that negative stains change under electron beam irradiation, for example, the volume of uranium acetate is reduced by up to 15%, which causes the protein surface movement (P.Un-win). Second, due to the negative staining solution and amino acid residue side chain gene chemistry. There will be positive or negative staining, making the image difficult to interpret (W.Chin). Finally, negatively stained samples can only get about 15 ~ 20A resolution. If not dyed, there is also a problem: the elephant contrast is very weak; there is a vacuum damage - the protein is damaged from drying due to moisture from the hydrated state to electron microscope vacuum; finally the radiation damage - the protein crystal is very sensitive to the electron beam, The protein crystals begin to break (Stenn) around the electron dose of le / A ~ 2. Such a dose is much smaller than the dose normally observed. In order to solve these problems, A. Klug et al. Used the following methods: imaging with phase contrast; replacing aqueous medium with glucose, which is good for vacuum protection because glucose is not volatile