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

1.构建了一个显微图像分析系统，该系统由倒置显微镜、显微操作系统、双视显微镜、配有像增强器的CCD摄像机、视频和图像处理器组成。利用这个系统，我们试图通过对pH2变化敏感的荧光图像来成像肌肉收缩系统中的机械力-化学耦合。我们合成了pH敏感的荧光染料SNAFL-Ph(SF-Ph)和snarf-Ph，它们可以特异性地与肌动蛋白细丝结合，并可以监测肌动蛋白分子周围的局部pH变化。利用这些染料和上述光学系统，我们可以获得随着Ca~(2+)和Gt~(2+)的激活而强度减弱的肌原纤维的荧光图像。我们还可以得到单个肌动蛋白细丝的荧光图像，其强度随着溶液pH的变化而可逆地变化。3.我们还构建了一种光镊子。利用该系统，我们可以捕获直径为1微米的塑料微珠，捕获力为70pN，并将微珠特定地附着在肌动蛋白细丝的带刺末端，从而测量出作用在肌动蛋白细丝上的滑动力和步长，前者最大可达3pN，后者约为10 nm。我们还测量了肌动蛋白细丝和HMM分子之间形成的单个刚性键的拉伸强度，平均为9.2pN。同时，我们可以得到该弹性体系的应力-应变关系，从该关系的最大斜率，我们可以估计单个HMM分子的平均弹性模数为0.5pN/nm。

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.