Crocker, John C
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Publication The Consensus Mechanics of Cultured Mammalian Cells(2006-07-05) Hoffman, Brenton D; Massiera, Gladys; Miranda, Kathleen A; Crocker, John CWhile understanding cells' responses to mechanical stimuli is seen as increasingly important for understanding cell biology, how to best measure, interpret and model cells' mechanical properties remains unclear. We determine the frequency-dependent shear modulus of cultured mammalian cells using four different methods, both novel and well established. This approach clarifies the effects of cytoskeletal heterogeneity, ATP-dependent processes and cell regional variations on the interpretation of such measurements. Our results clearly indicate two qualitatively similar but distinct mechanical responses, corresponding to the cortical and intracellular networks, each having an unusual, weak power-law form at low frequency. The two frequency dependent responses we observe are remarkably similar to those reported for a variety of cultured mammalian cells measured using different techniques, suggesting it is a useful consensus description. Finally, we discuss possible physical explanations for the observed mechanical response.Publication Microrheology of polyethylene oxide using diffusing wave spectroscopy and single scattering(2002-05-20) Dasgupta, Bivash R.; Tee, Shang-You; Crocker, John C; Frisken, B. J.; Weitz, D. A.Experiments investigating the local viscoelastic properties of a simple uncross-linked flexible polymer are performed on polyethylene oxide solutions in the semidilute regime using polystyrene beads of varying sizes and surface chemistry as probes. We measure the thermal motions of the beads to obtain the elastic and viscous moduli of our sample. Two different dynamic light scattering techniques, diffusing wave spectroscopy and quasielastic light scattering (QELS), are used to determine the dynamics of the probe particles. Diffusing wave spectroscopy probes the short time dynamics of the scatterers while QELS or single scattering measures the dynamics at larger times. This results in a larger frequency overlap of the data obtained from the microrheological techniques with the data obtained from the conventional bulk measurements. The moduli are estimated using a modified algebraic form of the generalized Stokes-Einstein equation. Comparison of microrheology with bulk measurements shows excellent similarity confirming the applicability of this method for simple, uncross-linked polymeric systems.Publication Stress-Dependent Elasticity of Composite Actin Networks as a Model for Cell Behavior(2006-03-03) Gardel, M. L; Nakamura, F.; Hartwig, J.; Crocker, John C; Stossel, T. P; Weitz, D. ANetworks of filamentous actin cross-linked with the actin-binding protein filamin A exhibit remarkable strain stiffening leading to an increase in differential elastic modulus by several orders of magnitude over the linear value. The variation of the frequency dependence of the differential elastic and loss moduli as a function of prestress is consistent with that observed in living cells, suggesting that cell elasticity is always measured in the nonlinear regime, and that prestress is an essential control parameter.Publication Microrheology of Entangled F-Actin Solutions(2003-10-07) Gardel, M. L.; Valentine, M. T.; Crocker, John C; Bausch, A. R.; Weitz, D. A.We measure the viscoelasticity of entangled F-actin over length scales between 1 and 100 µm using one- and two-particle microrheology, and directly identify two distinct microscopic contributions to the elasticity. Filament entanglements lead to a frequency-independent elastic modulus over an extended frequency range of 0:01–30 rad/ sec; this is probed with one-particle microrheology. Longitudinal fluctuations of the filaments increase the elastic modulus between 0.1 and 30 rad/ sec at length scales up to the filament persistence length; this is probed by two-particle microrheology.Publication Computational Analysis of Binary Segregation During Colloidal Crytallization with DNA-mediated Interactions(2010-06-17) Scarlett, Raynaldo T; Crocker, John C; Sinno, TalidA detailed computational study of compositional segregation during growth of colloidal binary solid-solution crystals is presented. Using a comprehensive set of Metropolis Monte Carlo simulations, we probe the influence of colloid size, interaction strength, and interaction range on the segregation process. The results are interpreted in terms of a simple, but descriptive mechanistic model that allows us to connect to studies of binary segregation in atomic systems. The validity of Metropolis Monte Carlo simulations for the nonequilibrium phenomena investigated in this work is established theoretically and by connections to Brownian dynamics and molecular dynamics simulations. It is demonstrated that standard Metropolis Monte Carlo, properly applied, can provide an efficient framework for studying many aspects of crystallization in colloidal systems.Publication Microrheology Probes Length Scale Dependent Rheology(2006-03-24) Liu, J.; Gardel, M. L.; Kroy, K.; Frey, E.; Hoffman, Brenton D; Crocker, John C; Bausch, A. R.; Weitz, D. A.We exploit the power of microrheology to measure the viscoelasticity of entangled F-actin solutions at different length scales from 1 to 100 µm over a wide frequency range. We compare the behavior of single probe-particle motion to that of the correlated motion of two particles. By varying the average length of the filaments, we identify fluctuations that dissipate diffusively over the filament length. These provide an important relaxation mechanism of the elasticity between 0.1 and 30 rad/sec.Publication Mechanics of Single Cells: Rheology, Time Dependence, and Fluctuations(2007-11-01) Massiera, Gladys; Van Citters, Kathleen M; Biancaniello, Paul L; Crocker, John CThe results of mechanical measurements on single cultured epithelial cells using both magnetic twisting cytometry (MTC) and laser tracking microrheology (LTM) are described. Our unique approach uses laser deflection for high-performance tracking of cell-adhered magnetic beads either in response to an oscillatory magnetic torque (MTC) or due to random Brownian or ATP-dependent forces (LTM). This approach is well suited for accurately determining the rheology of single cells, the study of temporal and cell-to-cell variations in the MTC signal amplitude, and assessing the statistical character of the tracers' random motion in detail. The temporal variation of the MTC rocking amplitude is surprisingly large and manifests as a frequency independent multiplicative factor having a 1/ƒ spectrum in living cells, which disappears upon ATP depletion. In the epithelial cells we study, random bead position fluctuations are Gaussian to the limits of detection both in the Brownian and ATP-dependent cases, unlike earlier studies on other cell types.Publication Line Optical Tweezers Instrument for Measuring Nanoscale Interactions and Kinetics(2006-11-15) Biancaniello, Paul L; Crocker, John CWe describe an optical tweezers instrument for measuring short-ranged colloidal interactions, based on a combination of a continuous wave line optical tweezers, high speed video microscopy, and laser illumination. Our implementation can measure the separation of two nearly contacting microspheres to better than 4 nm at rates in excess of 10 kHz. A simple image analysis algorithm allows us to sensibly remove effects from diffraction blurring and microsphere image overlap for separations ranging from contact to at least 100 nm. The result is a versatile instrument for measuring steric, chemical and single-molecular interactions and dynamics, with a force resolution significantly better than achievable with current atomic force microscopy. We demonstrate the effectiveness of the instrument with measurements of the pair interactions and dynamics of microspheres in the presence of transient molecular bridges of DNA or surfactant micelles.Publication Fragility and Mechanosensing in a Thermalized Cytoskeleton Model with Forced Protein Unfolding(2007-11-01) Hoffman, Brenton D; Massiera, Gladys; Crocker, John CWe describe a model of cytoskeletal mechanics based on the force-induced conformational change of protein cross-links in a stressed polymer network. Slow deformation of simulated networks containing cross-links that undergo repeated, serial domain unfolding leads to an unusual state — with many cross-links accumulating near the critical force for further unfolding. This state is robust to thermalization and does not occur in similar protein unbinding based simulations. Moreover, we note that the unusual configuration of near-critical protein cross-links in the fragile state provides a physical mechanism for the chemical transduction of cell-level mechanical strain and extra-cellular matrix stiffness.Publication Multiple Particle Tracking and Two-Point Microrheology in Cells(2007-01-01) Crocker, John C; Hoffman, Brenton DMechanical stress and stiffness are increasingly recognized to play important roles in numerous cell biological processes, notably cell differentiation and tissue morphogenesis. Little definite is known, however, about how stress propagates through different cell structures or how it is converted to biochemical signals via mechanotransduction, due in large part to the difficulty of interpreting many cell mechanics experiments. A newly developed technique, two-point microrheology (TPM), can provide highly interpretable, quantitative measurements of cells’ frequency-dependent shear moduli and spectra of their fluctuating intracellular stresses. TPM is a non-invasive method based on measuring the Brownian motion of large numbers of intracellular particles using multiple particle tracking. While requiring only hardware available in many cell biology laboratories–a phase microscope and digital video camera, as a statistical technique, it also requires the automated analysis of many thousands of micrographs. Here we describe in detail the algorithms and software tools used for such large-scale multiple particle tracking, as well as common sources of error and the microscopy methods needed to minimize them. Moreover, we describe the physical principles behind TPM and other passive microrheology methods, their limitations, and typical results for cultured epithelial cells.