Electron Microscopic Recording of Dynamic Structural Changes in Living Biomolecule

1.双极肌球蛋白细丝的制备：将肌球蛋白和肌球蛋白棒以1：10的比例混合，缓慢聚合成细丝，成功地制备出又长又粗的双极肌球蛋白细丝。所制备的双极丝长2-3微米，厚100-200 nm，适合于不染色条件下的电子显微镜记录。细丝上的肌球蛋白头部用金粒(直径15 nm)标记，每个金位标记的位置不随时间发生明显变化。当在没有ATP的情况下将F-肌动蛋白添加到肌丝中时，由于肌球蛋白头部和F-肌动蛋白之间形成了多个刚性连接，F-肌动蛋白细丝相互平行地附着在肌球蛋白细丝上。我们构建了软件程序来确定每个金粒子的质心位置。通过使用新的成像版系统，我们现在可以将电磁放大倍数从10,000倍降低到7,000倍，而不会降低以前的成像版系统所获得的特殊分辨率。结果表明，同一灯丝的图像可以被记录8次，而不会对空间造成严重的电子束损伤。我们在气体环境舱中一直使用的碳密封膜，很容易被激光或氙灯闪光损坏。我们尝试制备一种新型的SiO封闭膜，这种封闭膜可以抵抗激光或氙灯的闪光，从而使笼状化合物在闪光光解中快速释放活性化合物成为可能。然而，SiO膜很容易受到电子束的破坏。经过多次试验和失误，我们成功地制备了一种适合我们在气体环境舱中使用的SiO薄膜。

The results of the present research work are summarized as follows :1. Preparation of bipolar myosin filaments : We succeeded in preparing long and thick bipolar myosin filaments by mixing myosin and myosin rod at a ratio of 1 : 10, and slowly polymerizing them into filaments. The bipolar filaments thus prepared are 2-3um long and 100-200nm thick, and are suitable for electron microscopic recording under unstained conditions. The myosin head on the filament is marked with a gold particle (diameter, 15nm), and the position of each gold position marker does not change appreciably with time. When F-actin is added to the filaments in the absence of ATP, F-actin filaments are attached to the myosin filaments in parallel with each other due to multiple rigor linkage formation between the myosin head and F-actin.2. We constructed software programs to determine the centroid position of each gold particle. By the use of the new imaging plate system, we can now reduce EM magnification from 10,000X to 7,000X without reducing special resolution attained with the former imaging plate system. As the result, the image of the same filament can be recorded 8 times without any serious electron beam damage to the spacimen.3. The carbon sealing film, which we have been using in our gas environmental chamber, is readily damaged by laser or xenon lamp flash. We attempted to prepare a new sealing film with SiO, which is resistant to laser or xenon lamp flash, to make it possible to use caged compounds which rapidly release active compounds on photolysis with light flash. The SiO film is, however, readily damaged with electron beam. After a number of trials and errors we have succeeded in preparing a SiO film suitable for our use in the gas environmental chamber.