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

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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.