Biology Reference
In-Depth Information
z
-
x
or
z
-
y
can be minimized by aligning the particle of interest with the center of the
ring magnet.
6.1.3.7
Portable magnetic tweezers enables simultaneous imaging
and manipulation
In many cases, interpretation of microrheology data requires some knowledge of the
MT structure at the surface of the bead. In principle, the magnetic tweezers devices
described earlier could be mounted onto an inverted confocal microscope to enable
simultaneous imaging and microscope manipulation. In practice, this is a costly so-
lution, which requires a dedicated instrument with substantial structural modifica-
tions and custom software and therefore does not allow use of the sophisticated
core confocal imaging facilities available at many research institutions. As an alter-
native, we designed and constructed a custom portable magnetic tweezers device that
can be mounted onto any optical microscope (
Fig. 6.8
;
Yang, Lin, Meschewski,
Watson, & Valentine, 2011
).
In this design,
two small NdFeB magnets
(0.25
1 in.; Applied Magnets) are mounted onto a two-axis translation stage
and positioned near the focus of the objective lens in a manner that protects the im-
aging quality of the microscope. The distance between the magnets and sample can
be controllably varied, leading to application of controlled forces to small magnetic
particles at the sample plane.
For a typical measurement, the magnets are moved toward the sample until they
just touch the coverslip edge, to ensure application of the maximum force. To reduce
this force level, the magnets are moved away from the coverslip surface until the
separation distance exceeds
0.25
10 mm (at which point the force is nearly zero). The rate
ofmagnet retreat (and therefore the rate of force reduction) can be varied throughman-
ual control of the translation stage. In this “sideways-pulling” geometry, beads move
perpendicular to the optical axis (the “
x
” axis) when the force is on. We find that it is
most useful to obtain confocal images using both the fluorescence and the transmitted
light channels to visualize the MT network and beads, respectively. The magnetic
beads available commercially from Invitrogen are slightly autofluorescent under
561 nm excitation, which allows them to be observed in both channels. We find this
to be an advantage, since the bead-network interactions can be clearly observed.
In this case, particle position is determined using 3D centroid tracking.
6.1.4
Analysis of mechanical data
For microrheology measurements of homogeneous materials (i.e., materials in which
the bead radius is much larger than any other network length scale), it is possible to
determine the viscoelastic shear moduli directly through analysis of bead motion.
This is rarely the case for the MT networks in which the bead size is similar to
x
,
and the stiff filaments bend around the bead surface in a manner that does not lead
to uniform strain across the sample (e.g., the deformation is nonaffine). Thus, rather
than report elastic parameters in terms of modulus, which is a geometry-independent
materials property, we instead report network stiffness, which depends on both the
modulus and the structure of the network. Similarly, we report bead velocity rather
