Development and Application of the Method How to Combine Physiological Measurements with Electronmicroscopic Images at the Temporal Resolution of msec.

快速冷冻电子显微镜技术的最新进展大大提高了所能获得的图像的时间分辨率。已知时间分辨率至少为2毫秒。在本项目中，我们尝试开发一种新的冷冻系统，在该系统中，可以快速冷冻以下生物标本，同时测量它们在冷冻瞬间的生理状态。1.等张收缩条件下的甘油处理的兔骨骼肌：在缩短的肌肉中，即使在无负荷的情况下，大多数肌球蛋白头也沿着肌动蛋白丝的螺旋，肌球蛋白头部保持肌球蛋白亚基重复序列（143 A）的轴向间隔。2.等长收缩条件下的青蛙活体骨骼肌：骨骼肌的兴奋-收缩偶联伴随着肌浆网和横小管的显著结构变化，包括它们的连接的解离。3.暗适应和光适应的鱿鱼视网膜：暗适应的微绒毛的特征是具有轴丝，该轴丝与颗粒成分规则地结合并通过细长的股线与膜交联，并且光照射诱导颗粒成分从轴丝上解离和轴丝的断裂。鱿鱼巨轴突的轴膜由其底层的细胞骨架特化成两个域（微管和微管相关域）。这些“瞬时”观点将使我们更好地理解肌肉收缩、兴奋-收缩耦合、光感觉和膜兴奋的分子机制。

Recent progress in the rapid freezing electron microscope technique has greatly improved the temporal resolution of the images that can be obtained. The temporal resolution is known to be at least 2 msec. In this project, we have attempted to develop a new freezing system in which the following biological specimens can be rapidly frozen while measuring their physiological conditions at the very moment of freezing.1.Rabbit Glycerol-treated Skeletal Muscle under the Condition of Isotonic Contraction: In the shortening muscle, even in a unloaded condition, most myosin heads are situated along the helix of actin filaments, and that the myosin heads maintain the axial spacing of the myosin subunit repeat (143 A).2.Frog Living Skeletal Muscle under the Condition of Isometric Contraction: The excitation-contraction coupling in the skeletal muscle is accompanied by the remarkable structural changes of the sarcoplasmic reticulum and transverse-tubules including the dissociation of their junctions. 3.Dark-and Light-adapted Squid Retina: A dark-adapted microvillus is characterized unequivocally by an axial filament which is regularly associated with the granular components and cross-linked to the membrane through the slender strands, and that light-illimination induces the dissociation of granular components from the axial filaments and the fragmentation of the axial filaments.4.Squid Gilant Axon: The axolemma of the squid giant axon is specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons.These 'instantaneous' views will lead us to better understanding of the molecular mechanisms of muscle contraction, excitation-contraction coupling, light sensation, and membrane excitation.