Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod- tagged myosin V
Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod- tagged myosin V
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DOI：
10.1073/pnas.1118472109
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发表时间：
2012-04-03
影响因子：
11.1
通讯作者：
Yanagida, Toshio
中科院分区：
文献类型：
--
作者：
Ohmachi, Masashi;Komori, Yasunori;Yanagida, Toshio
单分子荧光偏振技术已被用于三维取向测量，以观察单分子的动力学性质。然而，只有少数技术可以同时测量3D方向和位置。此外，这些技术通常需要复杂的设备和繁琐的分析。我们开发了一套显微系统，并合成了具有线偏振的高荧光棒状量子点(Q棒)，用于同时测量单个荧光探针的位置和三维取向。这种光学装置根据偏振角度将来自探测器的荧光分成四个不同的光点，并将它们投射到ccd相机上。这些斑点用于确定2D位置和3D方向。在33ms的时间分辨率下，Q棒的定向精度优于10度。我们应用我们的显微镜和Q棒来同时测量肌球蛋白V沿肌动蛋白细丝的运动和围绕其自身轴的旋转，发现肌球蛋白V每一步旋转90度。根据这一结果，我们认为在双头束缚状态下，肌球蛋白V的脖子是相互垂直的，而在单头束缚状态下，分离的拖曳的肌球蛋白V头部部分地通过围绕自己的轴旋转杠杆臂而向前倾斜。这种显微系统应该适用于依赖于单分子取向动力学的广泛的动态生物过程。
Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10 degrees accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90 for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.