Biology Reference
In-Depth Information
introduced due to both the thermal fluctuations of the bead and the light scattering from
the dense MT network, which can degrade the diffraction of the bead used for 3D
tracking. For concentrated entangled networks and cross-linked networks, we estimate
our bead tracking accuracy to be
5 nm in all axes. For dilute entangled networks,
beads have higher mobility through the network, and our effective resolution is
degraded to
<
10-20 nm.
6.1.3.4
Fast force switching
Microrheology measurements frequently require fast switching of force amplitudes,
either to apply oscillatory stresses to a sample or to apply step stress pulses in a creep
measurement protocol. Fast force switching is easily implemented using electromag-
nets by rapidmodulation of the driving current. By contrast, for NdFeB-basedmagnetic
tweezers, force levels are modulated by the physical separation of the magnets and
sample. Although the incorporation of a fast linear motor tomove the magnets is trivial,
the ability to accurately track bead position during the repeated long-distance travel
of the magnet array is more challenging. To achieve accurate bead tracking during fast
force switching, a very stable long-distance travel motorized stage, a bright illumina-
tion source with an intensity that does not vary as the magnet array moves, and a
high-speed data acquisition system are required (
Lin & Valentine, 2012a
).
To ensure mechanical stability, the instrument is typically mounted onto an
air-cushioned optical table. A very stable, long-distance travel motorized stage
(M414.1PD, Physik Instrumente) is chosen to achieve high pulling force (200 N)
and high velocity (100 mm/s). For a typical microrheology experiment, the magnet ar-
ray is moved at most 15-20 mm to switch the applied force on or off. At maximum
motor travel speed, this transition would take
150-200 ms. An internal PID control-
ler, tuned to maximize acceleration, ensures
micron-scale repeatability. To damp vi-
brations, the motor is mounted onto a heavy column (XT95-1000, Thorlabs) filled with
1 mm steel shot, and the sample is mounted onto a custom-built heavy-duty platform.
To maintain tracking accuracy, the illumination intensity must be constant over the
entire magnet travel distance. To achieve this, we use a bright LED light source driven
by a stable, current-regulated power supply and focus the light onto the sample using a
custom-built optical collection system. This system must meet two critical demands.
First, as much LED light must be collected and focused onto the sample as is possible,
this will ensure that images can be collected at high frame rates (
100 fps) without
substantial contributions of shot noise. Second, the beam waist must be small enough
to pass through the magnet array, and the focal depth must be large enough to allow the
height of the magnet array to be adjusted without clipping the focused LED beam. To
achieve the desired force range for microrheology measurements, the focal depth must
be
>
1 m long. For
convenience, we mount the LED and illumination optics onto the same damped col-
umn that supports the long-distance travel motorized stage.
To acquire and process data quickly, we choose a high-resolution camera, fast
frame grabber, and high-performance computer workstation for real-time data anal-
ysis. We have found particular advantages with the use of a CMOS-based camera
(such as the Gazelle model, from Point Grey with a 2048
20-30 mm. In practice, this requires the illumination arm to be
1024 array of 5.5
m
m
