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Biophys J, July 2002, p. 491-501, Vol. 83, No. 1
and
Departments of *Biological Sciences, Physics, and
Applied Physics, Stanford University, Stanford,
California 94305-5020 USA
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.
Biophys J, July 2002, p. 491-501, Vol. 83, No. 1
© 2002 by the Biophysical Society 0006-3495/02/07/491/11 $2.00
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