An automated two-dimensional optical force clamp for single molecule studies. Author Matthew Lang, Charles Asbury, Joshua Shaevitz, Steven Block Publication Year 2002 Type Journal Article Abstract 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. Keywords Equipment Design, Biophysics, Calibration, Lasers, Kinesin, Microscopy, Video, Automation Journal Biophys J Volume 83 Issue 1 Pages 491-501 Date Published 07/2002 Alternate Journal Biophys. J. Google ScholarBibTeXEndNote X3 XML