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Because of its non-invasive nature, optical tweezers have emerged as a popular tool for the studies of complex fluids and
biological cells and tissues. The capabilities of optical tweezer-based experimental instruments continue to evolve for
better and broader applications, through new apparatus designs and integrations with microscopic imaging techniques. In
this paper, we present the design, calibration and applications of a powerful microrheometer that integrates a novel high
temporal and spatial resolution dual-beam oscillating optical tweezer-based cytorheometer (DOOTC) with spinning disk
confocal microscopy. The oscillating scheme detects the position of micron-size probe particles via a phase-sensitive
lock-in amplifier to greatly enhance sensitivity. The dual-beam scheme ensures that the cytorheometer is insensitive to
sample specimen background parameter variances, and thus enables the investigation of micromechanical properties of
biological samples, which are intrinsically inhomogeneous. The cytorheometer system is demonstrated to be capable of
measuring dynamic local mechanical moduli in the frequency range of 0.1-150 Hz at up to 2 data point per second and
with nanometer spatial resolutions, while visualizing and monitoring structural properties in situ. We report the results of
system applications in the studies of bovine skin gelatin gel, purified microtubule assemblies, and human alveolar
epithelial cells. The time evolution of the storage moduli G' and the loss moduli G'' of the gel is recorded for undisturbed
gel-forming process with high temporal resolution. The micromechanical modulus G* of polymerized
microtubule network as a function of frequency are shown to be both inhomogeneous and anisotropic consistent with
local structures revealed by confocal imaging. The mechanical properties of A549 human lung cells as a function of
temperature will be reported showing significant decrease in cell stiffness at higher temperature.
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