Simmons R M, Finer J T, Warrick H M, Kralik B, Chu S, Spudich J A
MRC Muscle and Motility Unit, King's College London, UK.
Adv Exp Med Biol. 1993;332:331-6; discussion 336-7. doi: 10.1007/978-1-4615-2872-2_32.
We have used an optical trap to measure or exert a force on single actin filaments via the attachment of polystyrene beads which were coated with NEM-modified HMM. In the simplest experiment, beads were attached to rhodamine phalloidin labelled actin filaments and observed to move on an HMM coated surface in the presence of ATP. Moving beads were steered into the vicinity of the trap using a PZT operated microscope stage. The minimum force needed to stop a moving bead was measured by lowering the trap strength until the bead resumed movement. By aligning the optical trap with the centre of a quadrant detector placed in an image plane of the microscope, it was possible to measure the force exerted on a filament by measuring the displacement of the bead position from the centre of the trap. In each of these experiments, the trap was calibrated by applying a Stokes force to a bead in free solution. The characteristics of the trap were studied, and the displacement of the bead from the centre of the trap was shown to be directly proportional to the applied force over a large part of the total range of the trap. The compliance of the trap could be substantially reduced by the use of feedback control to deflect the laser beam via an acousto-optic modulator. The advantages and limitations of this technique will be discussed.
我们使用光镊通过附着涂有NEM修饰的重酶解肌球蛋白（HMM）的聚苯乙烯珠子，来测量单个肌动蛋白丝或对其施加力。在最简单的实验中，将珠子附着在罗丹明鬼笔环肽标记的肌动蛋白丝上，并观察其在存在ATP的情况下在涂有HMM的表面上移动。使用由压电陶瓷（PZT）操作的显微镜载物台将移动的珠子引导到光镊附近。通过降低光镊强度直到珠子恢复移动，来测量使移动的珠子停止所需的最小力。通过将光镊与放置在显微镜图像平面中的象限探测器的中心对齐，可以通过测量珠子位置相对于光镊中心的位移来测量施加在细丝上的力。在每个实验中，通过对自由溶液中的珠子施加斯托克斯力来校准光镊。研究了光镊的特性，并且在光镊的大部分总范围内，珠子相对于光镊中心的位移与所施加的力成正比。通过使用反馈控制经由声光调制器使激光束偏转，可以大大降低光镊的柔度。将讨论该技术的优点和局限性。
