An automated two-dimensional optical force clamp for single molecule studies
An automated two-dimensional optical force clamp for single molecule studies
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DOI：
10.1016/s0006-3495(02)75185-0
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发表时间：
2002-07-01
影响因子：
3.4
通讯作者：
Block, SM
中科院分区：
文献类型：
--
作者：
Lang, MJ;Asbury, CL;Block, SM
我们构建了下一代光学捕获仪器来研究单个运动蛋白的运动性，例如沿着微管运动的驱动蛋白。该仪器可以作为二维力夹操作，向体外移动的电机涂层微珠施加固定大小和方向的负载。实验设计的灵活性和自动化是通过声光偏转器对陷阱位置和使用三维压电平台的样品位置进行计算机控制来实现的。每次测量之前都有一个初始化序列，其中包括使用光学力显微镜的变体（至+/-4 nm）调整相对于盖玻片的珠子高度，用于校准位置检测器响应的二维光栅扫描，以及相对于微管基底调整珠子横向位置（至+/-3 nm）。在电机驱动的移动过程中，捕获器和载物台都动态移动以施加恒定的力，同时将捕获的珠子保持在检测器的校准范围内。我们提供力钳操作的详细信息和显示驱动蛋白电机运动受对角线和前向负载影响的初步数据。
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.