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The Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) Twirling Filament Assay

机译：偏光全内反射荧光显微镜（polTIRFm）捻转丝试验

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Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes the twirling filament assay, so named because actin sometimes twirls about its own axis as it is translocated by myosin. A gliding filament assay is constructed in which a sparsely labeled actin filament (0.3% of the actin monomers contain 6′-iodoacetamidotetramethylrhodamine [IATR]) is translocated by a field of unlabeled myosin V fixed to the surface. The polTIRFM twirling assay differs from a standard gliding filament assay in that full filaments are not visible, but rather individual fluorophores are spaced along each filament. The goal is to investigate possible rotational motions of the actin filament about its axis (i.e., twirling) by measuring the spatial angle of the fluorescent probe as a function of time. Successful assays contain microscopic fields of approximately 50 isolated points of fluorescence that move across the field in the presence of ATP. Actin is usually translocated by more than one myosin molecule, depending on the filament length and the myosin surface density. Sparsely labeled filaments are required because the orientation of only one probe can be resolved at a time.
机译：偏振总内反射荧光显微镜（POLTIRFM）可用于检测单分子的空间取向和旋转动力学。 PolTirfm确定三维角度取向和荧光探针的摆动程度，荧光探针与感兴趣的大分子结合。该协议描述了旋转灯丝测定，所以命名，因为肌动蛋白有时会旋转其自身的轴，因为它被肌球蛋白易用。构建了滑动丝测定，其中稀疏标记的肌动蛋白丝（0.3％含有6'-碘乙酰氨基甲酰胺甲基溴胺[IATR]）由固定在表面上的未标记的肌蛋白V的领域。 PolTirfm旋转测定与标准滑动丝测定的不同之处在于该全部长丝不可见，但相当单独的荧光团沿每个长丝间隔开。目标是通过测量作为时间函数的荧光探针的空间角度来研究actin细丝围绕其轴线（即，旋转）的可能的旋转动作。成功的测定含有大约50个孤立的荧光点的微观场，其在ATP存在下穿过该领域。肌动蛋白通常由多于一种肌球蛋白分子易用，这取决于细丝长度和肌霉素表面密度。需要稀疏标记的细丝，因为一次只能解决一个探针的方向。

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