Lang Matthew J, Asbury Charles L, Shaevitz Joshua W, Block Steven M
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
Biophys J. 2002 Jul;83(1):491-501. doi: 10.1016/S0006-3495(02)75185-0.
We constructed a next-generation optical trapping instrument to study the motility of single motor proteins, such as kinesin moving along a microtubule. The instrument can be operated as a two-dimensional force clamp, applying loads of fixed magnitude and direction to motor-coated microscopic beads moving in vitro. Flexibility and automation in experimental design are achieved by computer control of both the trap position, via acousto-optic deflectors, and the sample position, using a three-dimensional piezo stage. Each measurement is preceded by an initialization sequence, which includes adjustment of bead height relative to the coverslip using a variant of optical force microscopy (to +/-4 nm), a two-dimensional raster scan to calibrate position detector response, and adjustment of bead lateral position relative to the microtubule substrate (to +/-3 nm). During motor-driven movement, both the trap and stage are moved dynamically to apply constant force while keeping the trapped bead within the calibrated range of the detector. We present details of force clamp operation and preliminary data showing kinesin motor movement subject to diagonal and forward loads.
我们构建了一台新一代光镊仪器，用于研究单个运动蛋白的运动，比如沿着微管移动的驱动蛋白。该仪器可作为二维力钳来操作，对在体外移动的包被有运动蛋白的微小珠子施加固定大小和方向的负载。通过使用声光偏转器对光阱位置以及使用三维压电平台对样品位置进行计算机控制，实现了实验设计的灵活性和自动化。每次测量之前都有一个初始化序列，包括使用光学力显微镜的一种变体将珠子相对于盖玻片的高度调整到（正负4纳米），进行二维光栅扫描以校准位置探测器的响应，以及将珠子相对于微管底物的横向位置调整到（正负3纳米）。在运动蛋白驱动的运动过程中，光阱和平台都会动态移动以施加恒定力，同时将被捕获的珠子保持在探测器的校准范围内。我们展示了力钳操作的细节以及初步数据，这些数据显示了驱动蛋白在对角负载和向前负载作用下的运动。
