Optical Tweezers Microrheology Research Articles

Switchable microscale stress response of actin-vimentin composites emerges from scale-dependent interactions.

The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments, including actin, microtubules, and intermediate filaments. Interactions between these distinct filaments give rise to emergent mechanical properties that are difficult to generate synthetically, and recent studies have made great strides in advancing our understanding of the mechanical interplay between actin and microtubule filaments. While intermediate filaments play critical roles in the stress response of cells, their effect on the rheological properties of the composite cytoskeleton remains poorly understood. Here, we use optical tweezers microrheology to measure the linear viscoelastic properties and nonlinear stress response of composites of actin and vimentin with varying molar ratios of actin to vimentin. We reveal a surprising, nearly opposite effect of actin-vimentin network mechanics compared to single-component networks in the linear versus nonlinear regimes. Namely, the linear elastic plateau modulus and zero-shear viscosity are markedly reduced in composites compared to single-component networks of actin or vimentin, whereas the initial response force and stiffness are maximized in composites versus single-component networks in the nonlinear regime. While these emergent trends are indicative of distinct interactions between actin and vimentin, nonlinear stiffening and longtime stress response appear to both be dictated primarily by actin, at odds with previous bulk rheology studies. We demonstrate that these complex, scale-dependent effects arise from the varied contributions of network density, filament stiffness, non-specific interactions, and poroelasticity to the mechanical response at different spatiotemporal scales. Cells may harness this complex behavior to facilitate distinct stress responses at different scales and in response to different stimuli to allow for their hallmark multifunctionality.

Micro-tensile rheology of fibrous gels quantifies strain-dependent anisotropy

Semiflexible fiber gels such as collagen and fibrin have unique nonlinear mechanical properties that play an important role in tissue morphogenesis, wound healing, and cancer metastasis. Optical tweezers microrheology has greatly contributed to the understanding of the mechanics of fibrous gels at the microscale, including its heterogeneity and anisotropy. However, the explicit relationship between micromechanical properties and gel deformation has been largely overlooked. We introduce a unique gel-stretching apparatus and employ it to study the relationship between microscale strain and stiffening in fibrin and collagen gels, focusing on the development of anisotropy in the gel. We find that gels stretched by as much as 15 % stiffen significantly both in parallel and perpendicular to the stretching axis, and that the parallel axis is 2–3 times stiffer than the transverse axis. We also measure the stiffening and anisotropy along bands of aligned fibers created by aggregates of cancer cells, and find similar effects as in gels stretched with the tensile apparatus. Our results illustrate that the extracellular microenvironment is highly sensitive to deformation, with implications for tissue homeostasis and pathology. Statement of significanceThe inherent fibrous architecture of the extracellular matrix (ECM) gives rise to unique strain-stiffening mechanics. The micromechanics of fibrous networks has been studied extensively, but the deformations involved in its stiffening at the microscale were not quantified. Here we introduce an apparatus that enables measuring the deformations in the gel as it is being stretched while simultaneously using optical tweezers to measure its microscale anisotropic stiffness. We reveal that fibrin and collagen both stiffen dramatically already at ∼10 % deformation, accompanied by the emergence of significant, yet moderate anisotropy. We measure similar stiffening and anisotropy in the matrix remodeled by the tensile apparatus to those found between cancer cell aggregates. Our results emphasize that small strains are enough to introduce substantial stiffening and anisotropy. These have been shown to result in directional cell migration and enhanced force propagation, and possibly control processes like morphogenesis and cancer metastasis.

Enzymatic cleaving of entangled DNA rings drives scale-dependent rheological trajectories.

DNA, which naturally occurs in linear, ring, and supercoiled topologies, frequently undergoes enzyme-driven topological conversion and fragmentation in vivo, enabling it to perform a variety of functions within the cell. In vitro, highly concentrated DNA polymers form entanglements that yield viscoelastic properties dependent on the topologies and lengths of the DNA. Enzyme-driven alterations of DNA size and shape therefore offer a means of designing active materials with programmable viscoelastic properties. Here, we incorporate multi-site restriction endonucleases into dense DNA solutions to linearize and fragment circular DNA molecules. We pair optical tweezers microrheology with differential dynamic microscopy and single-molecule tracking to measure the linear and nonlinear viscoelastic response and transport properties of entangled DNA solutions over a wide range of spatiotemporal scales throughout the course of enzymatic digestion. We show that, at short timescales, relative to the relaxation timescales of the polymers, digestion of these 'topologically-active' fluids initially causes an increase in elasticity and relaxation times followed by a gradual decrease. Conversely, for long timescales, linear viscoelastic moduli exhibit signatures of increasing elasticity. DNA diffusion, likewise, becomes increasingly slowed, in direct opposition to the short-time behavior. We hypothesize that this scale-dependent rheology arises from the population of small DNA fragments, which increases as digestion proceeds, driving self-association of larger fragments via depletion interactions, giving rise to slow relaxation modes of clusters of entangled chains, interspersed among shorter unentangled fragments. While these slow modes likely dominate at long times, they are presumably frozen out in the short-time limit, which instead probes the faster relaxation modes of the unentangled population.

Scale-dependent interactions enable emergent microrheological stress response of actin-vimentin composites.

The mechanical properties of the mammalian cell regulate many cellular functions and are largely dictated by the cytoskeleton, a composite network of protein filaments, including actin, microtubules, and intermediate filaments. Interactions between these distinct filaments give rise to emergent mechanical properties that are difficult to generate synthetically, and recent studies have made great strides in advancing our understanding of the mechanical interplay between actin and microtubule filaments. While intermediate filaments play critical roles in the stress response of cells, their effect on the rheological properties of the composite cytoskeleton remains poorly understood. Here, we use optical tweezers microrheology to measure the linear viscoelastic properties and nonlinear stress response of composites of actin and vimentin with varying molar ratios of actin to vimentin. We reveal a surprising, nearly opposite effect of actin-vimentin network mechanics compared to single-component networks in the linear versus nonlinear regimes. Namely, the linear elastic plateau modulus and zero-shear viscosity are markedly reduced in composites compared to single-component networks of actin or vimentin, whereas the initial response force and stiffness are maximized in composites versus single-component networks in the nonlinear regime. While these emergent trends are indicative of distinct interactions between actin and vimentin, nonlinear stiffening and long-time stress response appear to both be dictated primarily by actin, at odds with previous bulk rheology studies. We demonstrate that these complex, scale-dependent effects arise from the varied contributions of network density, filament stiffness, non-specific interactions, and poroelasticity to the mechanical response at different spatiotemporal scales. Cells may harness this complex behavior to facilitate distinct stress responses at different scales and in response to different stimuli to allow for their hallmark multifunctionality.

Microscale mechanics of actin-vimentin composite networks.

Most eukaryotic cells contain a complex network of cytoskeletal proteins comprised of filamentous actin, microtubule, and intermediate filaments. The ability of cells to resist mechanical deformation, transport cargo, and change shape during movement depends on these cytoskeletal proteins. Several previous studies have mainly focused on exploring the mechanics of in vitro reconstituted networks consisting of only one of these components, which may not be able to predict the properties of composite networks. Thus, investigating the interplay between the components as a composite is central to understanding the underlying biophysical principles. Here, we couple optical tweezers microrheology with confocal imaging to measure the viscoelastic response and structural dynamics of in vitro reconstituted filamentous actin (F-actin) and vimentin intermediate filaments (VIFs) composite networks by varying the relative concentration of the proteins. Our measurements shed new light on how VIFs and F-actin work synergistically during the various cellular processes.

Mechanics and stress propagation in non-equilibrium cytoskeleton composites

The cytoskeleton is able to access a wide range of material properties to facilitate cellular processes in a controlled and precise manner via active restructuring and reconfiguration of its constituents by ATP-consuming motor proteins. In vitro cytoskeleton composites have been shown to exhibit tunable mechanical properties, and the addition of force-generating motors to these composites induces non-equilibrium dynamics and restructuring. We characterize the effects of non-equilibrium activity on the mechanical properties of actin-microtubule composites driven by variable concentrations of kinesin motor clusters by performing optical tweezers microrheology experiments. We measure the nonlinear force response of the composites subject to cyclical strain at varied strain rates. Additionally, we measure stress propagation through the composites due to locally imposed strain using differential dynamic microscopy (DDM). We uncover a transition from dissipative to stiffening force response of the composites at mesoscopic lengthscales, with a non-monotonic dependence on kinesin concentration. Our work sheds light on how active restructuring by motor proteins tunes the material response of the cytoskeleton to local stresses, enabling diverse processes such as shape change, motility, and repair.

Optical tweezers and DDM characterize time-varying strain fields in kinesin-driven cytoskeleton composites.

The cytoskeleton continuously self-generates forces and reconfigures itself - in part by motor proteins pushing and pulling on the comprising filaments - to enable diverse mechanical responses to local stresses and strains. We previously showed that kinesin-microtubule interactions lead to dynamics ranging from uncorrelated isotropic fluctuations, to mesoscale restructuring, to bulk flow of actin-microtubule composites. These non-equilibrium dynamics mediate varying degrees of de-mixing, clustering and bundling of originally interpenetrating networks of actin and microtubules. Here, we combine optical tweezers microrheology and fluorescence microscopy to visualize the dynamical response and strain field of composites subject to cyclic mesoscale straining. To characterize the time-varying filament deformations and stress propagation we use space- and time-resolved differential dynamic microscopy (DDM) for quantifying the strain alignment, speed, periodicity, and dissipation of the network dynamics. We show that intermediate concentrations of kinesin motors lead to the most pronounced strain alignment and stress propagation while both low and high motor concentrations lead to more rapidly decaying response. Composite dynamics also exhibit similar emergent non-monotonic dependences on strain speed owing to the different relaxation mechanisms available to structurally-evolving composites.

Nonlinear Microscale Mechanics of Actin Networks Governed by Coupling of Filament Crosslinking and Stabilization.

Actin plays a vital role in maintaining the stability and rigidity of biological cells while allowing for cell motility and shape change. The semiflexible nature of actin filaments-along with the myriad actin-binding proteins (ABPs) that serve to crosslink, bundle, and stabilize filaments-are central to this multifunctionality. The effect of ABPs on the structural and mechanical properties of actin networks has been the topic of fervent investigation over the past few decades. Yet, the combined impact of filament stabilization, stiffening and crosslinking via ABPs on the mechanical response of actin networks has yet to be explored. Here, we perform optical tweezers microrheology measurements to characterize the nonlinear force response and relaxation dynamics of actin networks in the presence of varying concentrations of α-actinin, which transiently crosslinks actin filaments, and phalloidin, which stabilizes filamentous actin and increases its persistence length. We show that crosslinking and stabilization can act both synergistically and antagonistically to tune the network resistance to nonlinear straining. For example, phalloidin stabilization leads to enhanced elastic response and reduced dissipation at large strains and timescales, while the initial microscale force response is reduced compared to networks without phalloidin. Moreover, we find that stabilization switches this initial response from that of stress stiffening to softening despite the increased filament stiffness that phalloidin confers. Finally, we show that both crosslinking and stabilization are necessary to elicit these emergent features, while the effect of stabilization on networks without crosslinkers is much more subdued. We suggest that these intriguing mechanical properties arise from the competition and cooperation between filament connectivity, bundling, and rigidification, shedding light on how ABPs with distinct roles can act in concert to mediate diverse mechanical properties of the cytoskeleton and bio-inspired polymeric materials.

Polymer threadings and rigidity dictate the viscoelasticity of entangled ring-linear blends and their composites with rigid rod microtubules

Mixtures of polymers of varying topologies and stiffnesses display complex emergent rheological properties that often cannot be predicted from their single-component counterparts. For example, entangled blends of ring and linear polymers have been shown to exhibit enhanced shear thinning and viscosity, as well as prolonged relaxation timescales, compared to pure solutions of rings or linear chains. These emergent properties arise in part from the synergistic threading of rings by linear polymers. Topology has also been shown to play an important role in composites of flexible (e.g., DNA) and stiff (e.g., microtubules) polymers, whereby rings promote mixing while linear polymers induce demixing and flocculation of stiff polymers, with these topology-dependent interactions giving rise to highly distinct rheological signatures. To shed light on these intriguing phenomena, we use optical tweezers microrheology to measure the linear and nonlinear rheological properties of entangled ring-linear DNA blends and their composites with rigid microtubules. We show that linear viscoelasticity is primarily dictated by microtubules at lower frequencies, but their contributions become frozen out at frequencies above the DNA entanglement rate. In the nonlinear regime, we reveal that mechanical response features, such as shear thinning and stress softening, are mediated by entropic stretching, threading, and flow alignment of entangled DNA, as well as forced dethreading, disentanglement, and clustering. The contributions of each of these mechanisms depend on the strain rate as well as the entanglement density and stiffness of the polymers, leading to nonmonotonic rate dependences of mechanical properties that are most pronounced for highly concentrated ring-linear blends rather than DNA-microtubule composites.

DNA Conformation Dictates Strength and Flocculation in DNA-Microtubule Composites.

Polymer topology has been shown to play a key role in tuning the dynamics of complex fluids and gels. At the same time, polymer composites, ubiquitous in everyday life, have been shown to exhibit emergent desirable mechanical properties not attainable in single-species systems. Yet, how topology impacts the dynamics and structure of polymer composites remains poorly understood. Here, we create composites of rigid rods (microtubules) polymerized within entangled solutions of flexible linear and ring polymers (DNA) of equal length. We couple optical tweezers microrheology with confocal microscopy and scaled particle theory to show that composites with linear DNA exhibit a strongly nonmonotonic dependence of elasticity and stiffness on microtubule concentration due to depletion-driven polymerization and flocculation of microtubules. In contrast, composites containing ring DNA show a much more modest monotonic increase in elastic strength with microtubule concentration, which we demonstrate arises from the decreased conformational size and increased miscibility of rings.

Salt-Mediated Stiffening, Destruction, and Resculpting of Actomyosin Network

Cells dynamically change their viscoelastic properties by restructuring networks of actin filaments in the cytoskeleton, enabling diverse mechanical processes such as mobility and apoptosis. This restructuring is modulated, in part, by actin-binding proteins, such as myosin II, as well as counterions such as Mg2+ and K+. While high concentrations of Mg2+ can induce bundling and crosslinking of actin filaments, high concentrations of K+ destabilize myosin II minifilaments necessary to crosslink actin filaments. Here, we elucidate how the mechanics and structure of actomyosin networks evolve under competing effects of varying Mg2+ and K+ concentrations. Specifically, we couple microfluidics with optical tweezers microrheology to measure the time-varying linear viscoelastic moduli of actin networks crosslinked via myosin II as we cycle between low and high Mg2+ and K+ concentrations. Our complementary confocal imaging experiments correlate the time-varying viscoelastic properties with salt-mediated structural evolution. We find that the elastic modulus displays an intriguing non-monotonic time dependence in high-salt conditions, that correlates with structural changes, and that this process is irreversible, with the network evolving to a new steady-state as Mg2+ and K+ decrease back to their initial concentrations.

Motor-Driven Restructuring of Cytoskeleton Composites Leads to Tunable Time-Varying Elasticity.

The composite cytoskeleton, comprising interacting networks of semiflexible actin and rigid microtubules, generates forces and restructures by using motor proteins such as myosins to enable key processes including cell motility and mitosis. Yet, how motor-driven activity alters the mechanics of cytoskeleton composites remains an open challenge. Here, we perform optical tweezers microrheology and confocal imaging of composites with varying actin-tubulin molar percentages (25-75, 50-50, and 75-25), driven by light-activated myosin II motors, to show that motor activity increases the elastic plateau modulus by over 2 orders of magnitude by active restructuring of both actin and microtubules that persists for hours after motor activation has ceased. Nonlinear microrheology measurements show that motor-driven restructuring increases the force response and stiffness and suppresses actin bending. The 50-50 composite exhibits the most dramatic mechanical response to motor activity due to the synergistic effects of added stiffness from the microtubules and sufficient motor substrate for pronounced activity.

Viscoelastic properties of ring-linear DNA blends exhibit nonmonotonic dependence on blend composition

Entangled ring polymers, along with blends of ring and linear polymers, continue to be a topic of great interest and debate due to the conflicting experimental results in the literature as well as the difficulty of producing entangled synthetic rings devoid of linear contaminants. Here, we create blended solutions of entangled ring and linear DNA with varying mass fractions of linear DNA. We use optical tweezers microrheology to measure the linear and nonlinear viscoelastic response of these blends. Our measurements reveal a strong non-monotonic dependence of linear viscoelastic properties on linear DNA fraction, with a pronounced maximum when the mass fraction of rings and linear chains are comparable, suggestive of pervasive threading of rings by linear chains. We observe a similar non-monotonicity in the nonlinear regime; however, a comparatively higher fraction of linear chains (0.5-0.7) is required for a substantial increase in resisitive force and slowing of relaxation dynamics to emerge. This nonlinear response also appears to be rate dependent, which we argue arises from force-induced de-threading of rings at high strain rates. Our results fill a longstanding gap in knowledge regarding the microrheology and nonlinear response of ring-linear polymer blends. Moreover, the uniquely strong mechanical response that ring-linear blends exhibit, along with the ability to finely tune these blends by varying the blend composition, provides new materials design principles.

Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule composites

The cytoskeleton precisely tunes its mechanics by altering interactions between semiflexible actin filaments, rigid microtubules, and crosslinking proteins. We use optical tweezers microrheology and confocal microscopy to characterize how varying crosslinking motifs impact the mesoscale mechanics and mobility of actin-microtubule composites. We show that, upon subtle changes in crosslinking patterns, composites can exhibit two distinct classes of force response – primarily elastic versus more viscous. For example, a composite in which actin and microtubules are crosslinked to each other but not to themselves is markedly more elastic than one in which both filaments are independently crosslinked. Notably, this distinction only emerges at mesoscopic scales in response to nonlinear forcing, whereas varying crosslinking motifs have little impact on the microscale mechanics and mobility. Our unexpected scale-dependent results not only inform the physics underlying key cytoskeleton processes and structures, but, more generally, provide valuable perspective to materials engineering endeavors focused on polymer composites.

Optical Tweezers Microrheology Maps the Dynamics of Strain-Induced Local Inhomogeneities in Entangled Polymers.

Optical tweezers microrheology (OTM) offers a powerful approach to probe the nonlinear response of complex soft matter systems, such as networks of entangled polymers, over wide-ranging spatiotemporal scales. OTM can also uniquely characterize the microstructural dynamics that lead to the intriguing nonlinear rheological properties that these systems exhibit. However, the strain in OTM measurements, applied by optically forcing a microprobe through the material, induces network inhomogeneities in and around the strain path, and the resultant flow field complicates the measured response of the system. Through a robust set of custom-designed OTM protocols, coupled with modeling and analytical calculations, we characterize the time-varying inhomogeneity fields induced by OTM measurements. We show that homogenization following strain does not interfere with the intrinsic stress relaxation dynamics of the system, rather it manifests as an independent component in the stress decay, even in highly nonlinear regimes such as with the microrheological large-amplitude-oscillatory-shear (MLAOS) protocols we introduce. Our specific results show that Rouse-like elastic retraction, rather than disentanglement and disengagement, dominates the nonlinear stress relaxation of entangled polymers at micro- and mesoscales. Thus, our study opens up possibilities of performing precision nonlinear microrheological measurements, such as MLAOS, on a wide range of complex macromolecular systems.

Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites.

The cytoskeleton is able to precisely tune its structure and mechanics through interactions between semiflexible actin filaments, rigid microtubules and a suite of crosslinker proteins. However, the role that each of these components, as well as the interactions between them, plays in the dynamics of the composite cytoskeleton remains an open question. Here, we use optical tweezers microrheology and fluorescence confocal microscopy to reveal the surprising ways in which actin crosslinking tunes the viscoelasticity and mobility of actin-microtubule composites from steady-state to the highly nonlinear regime. While previous studies have shown that increasing crosslinking in actin networks increases elasticity and stiffness, we instead find that composite stiffness displays a striking non-monotonic dependence on actin crosslinking - first increasing then decreasing to a response similar to or even lower than un-linked composites. We further show that actin crosslinking has an unexpectedly strong impact on the mobility of microtubules; and it is in fact the microtubule mobility - dictated by crosslinker-driven rearrangements of actin filaments - that controls composite stiffness. This result is at odds with conventional thought that actin mobility drives cytoskeleton mechanics. More generally, our results demonstrate that - when crosslinking composite materials to confer strength and resilience - more is not always better.

Maximally stiffening composites require maximally coupled rather than maximally entangled polymer species.

Polymer composites are ideal candidates for next generation biomimetic soft materials because of their exquisite bottom-up designability. However, the richness of behaviours comes at a price: the need for precise and extensive characterisation of material properties over a highly-dimensional parameter space, as well as a quantitative understanding of the physical principles underlying desirable features. Here we couple large-scale Molecular Dynamics simulations with optical tweezers microrheology to characterise the viscoelastic response of DNA-actin composites. We discover that the previously observed non-monotonic stress-stiffening of these composites is robust, yet tunable, in a broad range of the parameter space that spans two orders of magnitude in DNA length. Importantly, we discover that the most pronounced stiffening is achieved when the species are maximally coupled, i.e., have similar number of entanglements, and not when the number of entanglements per DNA chain is largest. We further report novel dynamical oscillations of the microstructure of the composites, alternating between mixed and bundled phases, opening the door to future investigations. The generic nature of our system renders our results applicable to the behaviour of a broad class of polymer composites.

Triggered disassembly and reassembly of actin networks induces rigidity phase transitions.

Non-equilibrium soft materials, such as networks of actin proteins, have been intensely investigated over the past decade due to their promise for designing smart materials and understanding cell mechanics. However, current methods are unable to measure the time-dependent mechanics of such systems or map mechanics to the corresponding dynamic macromolecular properties. Here, we present an experimental approach that combines time-resolved optical tweezers microrheology with diffusion-controlled microfluidics to measure the time-evolution of microscale mechanical properties of dynamic systems during triggered activity. We use these methods to measure the viscoelastic moduli of entangled and crosslinked actin networks during chemically-triggered depolymerization and repolymerization of actin filaments. During disassembly, we find that the moduli exhibit two distinct exponential decays, with experimental time constants of ∼169 min and ∼47 min. Conversely, during reassembly, measured moduli initially exhibit power-law increase with time, after which steady-state values are achieved. We develop toy mathematical models that couple the time-evolution of filament lengths with rigidity percolation theory to shed light onto the molecular mechanisms underlying the observed mechanical transitions. The models suggest that these two distinct behaviors both arise from phase transitions between a rigidly percolated network and a non-rigid regime. Our approach and collective results can inform the general principles underlying the mechanics of a large class of dynamic, non-equilibrium systems and materials of current interest.

Co-Entangled Actin-Microtubule Composites Exhibit Tunable Stiffness and Power-Law Stress Relaxation

We use optical tweezers microrheology and fluorescence microscopy to characterize the nonlinear mesoscale mechanics and mobility of in vitro co-entangled actin-microtubule composites. We create a suite of randomly oriented, well-mixed networks of actin and microtubules by co-polymerizing varying ratios of actin and tubulin in situ. To perturb each composite far from equilibrium, we use optical tweezers to displace an embedded microsphere a distance greater than the lengths of the filaments at a speed much faster than their intrinsic relaxation rates. We simultaneously measure the force the filaments exert on the bead and the subsequent force relaxation. We find that the presence of a large fraction of microtubules (>0.7) is needed to substantially increase the measured force, which is accompanied by large heterogeneities in force response. Actin minimizes these heterogeneities by reducing the mesh size of the composites and supporting microtubules against buckling. Composites also undergo a sharp transition from strain softening to stiffening when the fraction of microtubules (ϕT) exceeds 0.5, which we show arises from faster poroelastic relaxation and suppressed actin bending fluctuations. The force after bead displacement relaxes via power-law decay after an initial period of minimal relaxation. The short-time relaxation profiles (t < 0.06 s) arise from poroelastic and bending contributions, whereas the long-time power-law relaxation is indicative of filaments reptating out of deformed entanglement constraints. The scaling exponents for the long-time relaxation exhibit a nonmonotonic dependence on ϕT, reaching a maximum for equimolar composites (ϕT = 0.5), suggesting that reptation is fastest in ϕT = 0.5 composites. Corresponding mobility measurements of steady-state actin and microtubules show that both filaments are indeed the most mobile in ϕT = 0.5 composites. This nonmonotonic dependence of mobility on ϕT demonstrates the important interplay between mesh size and filament rigidity in polymer networks and highlights the surprising emergent properties that can arise in composites.

Optical Tweezers Microrheology: From the Basics to Advanced Techniques and Applications.

Over the past few decades, microrheology has emerged as a widely used technique to measure the mechanical properties of soft viscoelastic materials. Optical tweezers offer a powerful platform for performing microrheology measurements and can measure rheological properties at the level of single molecules out to near macroscopic scales. Unlike passive microrheology methods, which use diffusing microspheres to extract rheological properties, optical tweezers can probe the nonlinear viscoelastic response, and measure the space- and time-dependent rheological properties of heterogeneous, nonequilibrium materials. In this Viewpoint, I describe the basic principles underlying optical tweezers microrheology, the instrumentation and material requirements, and key applications to widely studied soft biological materials. I also describe several sophisticated approaches that include coupling optical tweezers to fluorescence microscopy and microfluidics. The described techniques can robustly characterize noncontinuum mechanics, nonlinear mechanical responses, strain-field heterogeneities, stress propagation, force relaxation dynamics, and time-dependent mechanics of active materials.