Microscopic image analysis of mechanochemical coupling in contractile system of muscle.

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1.We constructed a microscopic image-analysis system which consists of an inverted microscope, microscopic manipulation system, Double-view (W) microscopy, a CCD camera equipped with an image intensifier, a video and image-processor. Using this sistem, we tried to image a mechano-chemical coupling in the contractile system of muscle through the fluorescent image which is sensitive to the change of pH.2.We synthesized pH sensitive fluorescent dyes called SNAFL-phalloidin (SF-Ph) and SNARF-Ph which specifically bind to actin filaments and can monitor the local pH change around the actin molecules. Using these dyes and the above optical system, we could obtain the fluorescence image of myofibrils of which intensity decreased accompanying the activation with Ca^<2+>. We could also obtain the fluorescence image of single actin filaments of which intensity reversibly changed with the change of pH of solution.3.We also constructed an optical tweezers. Using this system, we could trap a plastic bead of 1mum in diameter with a trapping force of 70 pN.The bead was specifically attached to the barbed end of an actin filament so that the sliding force exerted on the actin filament and a step size were measured ; the former was 3 pN at maximum and the latter was about 10nm. We also measured the tensile strength of single rigor bond formed between an actin filament and a HMM molecule ; it was 9.2 pN on the average. At the same time, we could obtain the stress-strain relation of this elastic system ; from the steepest slope of this relation, we could estimate the elastic modulus of single HMM molecules to be 0.5 pN/nm on the average.