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Over the past few decades, “microrheology” has emerged as a widely-used technique to measure the mechanical properties of soft viscoelastic materials, many of which are derived from biology. These methods offer an alternative to conventional “bulk” rheology, which can require prohibitively large sample volumes, can damage fragile materials, and cannot resolve microscale heterogeneities or deformations of individual macromolecules. Optical tweezers offer a powerful platform for performing microrheology measurements that can uniquely measure rheological properties at the level of single molecules out to near macroscopic scales. Unlike passive microrheology methods, which use diffusing microspheres to extract steady-state rheological properties, optical tweezers can access the nonlinear mechanical response of materials and measure the space- and time-dependent rheological properties of heterogeneous, non-equilibrium materials. I will describe the basic principles underlying how optical tweezers can be used to perform microrheology measurements. I will discuss instrumentation requirements, benefits over other methods, and material systems that are most amenable to the method. I will also describe several novel approaches that include coupling optical tweezers with fluorescence microscopy and microfluidics, and using single molecules as stress and strain probes. These novel configurations can characterize non-continuum mechanical properties, nonlinear viscoelasticity, strain-field heterogeneities, stress propagation, force relaxation dynamics, and time-dependent active matter mechanics. Finally, I will show examples of applications of these methods to widely-studied soft biological materials including entangled DNA, cytoskeleton protein networks, and mucus.
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