A high-resolution magnetic tweezer for
Kipom Kim1and Omar A. Saleh1,2,*
1Materials Department and2Biomolecular Science and Engineering Program, University of California,
Santa Barbara, CA 93106, USA
Received May 1, 2009; Revised July 26, 2009; Accepted August 17, 2009
manipulation instruments that utilize a magnetic
field to apply force to a biomolecule-tethered
magnetic bead while using optical bead tracking
to measure the biomolecule’s extension. While
relatively simple to set up, prior MT implementa-
tions have lacked the resolution necessary to
observe sub-nanometer biomolecular configuration
changes. Here,we demonstrate
interference technique for bead tracking, and show
that it has much better resolution than traditional
resolution by fabricating optical coatings on all
reflecting surfaces that optimize the intensity and
implement feedback control of the focal position
to remove drift. To test the system, we measure
the length change of a DNA hairpin as it undergoes
a folding/unfolding transition.
Over the past 10 years, a variety of experimental
techniques have been developed that allow the mechanical
manipulation of a single biological molecule and the
sensing of its interactions with other biomolecules (1–3).
These single-molecule measurements are extraordinarily
sensitive, as they are capable of applying piconewton-
scale forces and measuring displacements of nanometers,
allowing them to provide direct data on the molecular-
scale workings of biological systems. Importantly, these
data are quantitative, and can thus be directly compared
to other information, such as crystallographic structures
or biochemical / biophysical theories, in order to attain
a complete understanding of biomolecular behavior.
The magnetic tweezer (MT) is a simple and stable tool
to stretch and twist biomolecules and to measure their
extension over time (4–8). It is based on the manipulation,
using magnetic fields, of a paramagnetic bead (1–5mm in
diameter) that is tethered to a glass surface through a
single biomolecule. The field applies a force to the bead
that stretches or twists the molecule. In a typical
experiment, a physical change to the tethered biomolecule
causes its extension to increase or decrease, raising or
lowering the vertical position of the bead. Traditionally,
bead height is measured by analyzing the bead’s
diffraction image when viewed in a transmitted light
geometry (9,10). Thus, the MT’s capability to measure
physical changes to the biomolecule depends on the
ability to relate variations in the diffraction image to the
position of the bead.
The resolution of any single-molecule technique is
impaired by two sources of noise: the Brownian motion
of the bead, which can be reduced by using small beads,
high forces, and short tethers, and the intrinsic resolution
of the instrument, which depends on the sensing technique
diffraction-based MT setups, the intrinsic resolution is
limited to ?1nm (10), which suffers in comparison to
high-resolution optical tweezer methods (11). This low-
resolution originates from the relatively small lateral
motion of the diffraction interference fringes with bead
height. Limited resolution is acceptable in the low-force
regime, where Brownian motion of the tethered bead
dominates the experimental noise. However, when using
high forces, Brownian motion is reduced and the intrinsic
resolution becomes limiting.
To overcome this limit, we have adapted Reflection
Interference Contrast Microscopy (RICM) to the MT.
interference pattern between rays reflecting from a glass
surface and those reflecting from an object in solution
located near to the surface (12,13). Bead height is
calculated by comparing the
pattern to a theoretical model that predicts the fringe
shape based on the optical geometry (13). Importantly,
the interference fringes from RICM have a much larger
variation with bead height than those from diffraction.
Indeed, prior applications of RICM have indicated
sub-nanometer accuracy in measured bead height is
illumination creates an
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Published online 3 September 2009 Nucleic Acids Research, 2009, Vol. 37, No. 20e136
? The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
resolving step-like extension changes requires an SNR >4
(22); furthermore, the choice of window size will be
determined by the kinetics of the studied extension-
changing process. Thus Figure 7b indicates that, for a
1738bp tether under 23pN of force, 1nm steps can be
resolved by RICM if they occur more slowly than
We have demonstrated a high resolution MT based on
RICM with a greater than 10-fold resolution increase
over the traditional diffraction method. This improvement
was enabled by the intrinsically higher sensitivity of the
RICM fringes, the inclusion of thin films that enhanced
the quality of the RICM interferograms, and the use of an
active stabilization routine. We used a biomolecular
calibration based on DNA hairpin unfolding to show
that the RICM measurement of the length change is
accurate. Our results show that the RICM-based MT
system is well suited to precision measurements of
configuration-changing biomolecular activity. We expect
this system will provide a simple and powerful tool for the
investigation of the physical properties of biological
Supplementary Data are available at NAR Online.
The authors thank N. Ribeck for comments.
1R21GM079584-01A1]. A portion of this work was
done in the UCSB nanofabrication facility, part of the
Nanotechnology Infrastructure Network. Funding for
open access charge: National Institutes of Health [grant
Conflict of interest statement. None declared.
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