Intracellular nonequilibrium fluctuating stresses indicate how nonlinear cellular mechanical properties adapt to microenvironmental rigidity.

Living cells are known to be in thermodynamically nonequilibrium, which is largely brought about by intracellular molecular motors. The motors consume chemical energies to generate stresses and reorganize the cytoskeleton for the cell to move and divide. However, since there has been a lack of direct measurements characterizing intracellular stresses, questions remained unanswered on the intricacies of how cells use such stresses to regulate their internal mechanical integrity in different microenvironments. This report describes a new experimental approach by which we reveal an environmental rigidity-dependent intracellular stiffness that increases with intracellular stress - a revelation obtained, surprisingly, from a correlation between the fluctuations in cellular stiffness and that of intracellular stresses. More surprisingly, by varying two distinct parameters, environmental rigidity and motor protein activities, we observe that the stiffness-stress relationship follows the same curve. This finding provides some insight into the intricacies by suggesting that cells can regulate their responses to their mechanical microenvironment by adjusting their intracellular stress.

Frequency Response of Induced-Charge Electrophoretic Metallic Janus Particles.

The ability to manipulate and control active microparticles is essential for designing microrobots for applications. This paper describes the use of electric and magnetic fields to control the direction and speed of induced-charge electrophoresis (ICEP) driven metallic Janus microrobots. A direct current (DC) magnetic field applied in the direction perpendicular to the electric field maintains the linear movement of particles in a 2D plane. Phoretic force spectroscopy (PFS), a phase-sensitive detection method to detect the motions of phoretic particles, is used to characterize the frequency-dependent phoretic mobility and drag coefficient of the phoretic force. When the electric field is scanned over a frequency range of 1 kHz-1 MHz, the Janus particles exhibit an ICEP direction reversal at a crossover frequency at ~30 kH., Below this crossover frequency, the particle moves in a direction towards the dielectric side of the particle, and above this frequency, the particle moves towards the metallic side. The ICEP phoretic drag coefficient measured by PFS is found to be similar to that of the Stokes drag. Further investigation is required to study microscopic interpretations of the frequency at which ICEP mobility switched signs and the reason why the magnitudes of the forward and reversed modes of ICEP are so different.

Dielectrophoresis and colloidal phase transitions for ultra-broadband optical limiting.

We demonstrate wavelength-independent optical limiting based on colloidal phase transitions induced by the dielectrophoretic force from focused electromagnetic radiation. The focused radiation acts as an optical trap that increases the particle density. The increased density then leads to a colloidal gas-solid phase transition and an aggregate that effectively blocks the incoming radiation when it passes a threshold power. The process is reversible, with the colloidal particles returning to a homogenous distribution after the incoming radiation is removed. We demonstrate the effect using polystyrene nanoparticles mixed with pluronics and polyethylene glycol polymers in low-concentration KCl salt solutions. We observe the light-induced phase separation under confocal fluorescent microscope, and we provide a proof-of-principle demonstration of optical limiting using a 100 µm thick colloid cell.

Regulating substrate mechanics to achieve odontogenic differentiation for dental pulp stem cells on TiO2 filled and unfilled polyisoprene.

We have shown that materials other than hydrogels commonly used in tissue engineering can be effective in enabling differentiation of dental pulp stem cells (DPSC). Here we demonstrate that a hydrophobic elastomer, polyisoprene (PI), a component of Gutta-percha, normally used to obturate the tooth canal, can also be used to initiate differentiation of the pulp. We showed that PI substrates without additional coating promote cell adhesion and differentiation, while their moduli can be easily adjusted either by varying the coating thickness or incorporation of inorganic particles. DPSC plated on those PI substrates were shown, using SPM and hysitron indentation, to adjust their moduli to conform to differentially small changes in the substrate modulus. In addition, optical tweezers were used to separately measure the membrane and cytoplasm moduli of DPSC, with and without Rho kinase inhibitor. The results indicated that the changes in modulus were attributed predominantly to changes within the cytoplasm, rather than the cell membrane. CLSM was used to identify cell morphology. Differentiation, as determined by qRT-PCR, of the upregulation of OCN, and COL1α1 as well as biomineralization, characterized by SEM/EDAX, was observed on hard PI substrates in the absence of induction factors, i.e. dexamethasone, with moduli 3-4 MPa, regardless of preparation. SEM showed that even though biomineralization was deposited on both spun cast thin PI and filled thick PI substrates, the minerals were aggregated into large clusters on thin PI, and uniformly distributed on filled thick PI, where it was templated within banded collagen fibers. STATEMENT OF SIGNIFICANCE: This manuscript demonstrates the potential of polyisoprene (PI), an elastomeric polymer, for use in tissue engineering. We show how dental pulp stem cells adjust their moduli continuously to match infinitesimally small changes in substrate mechanics, till a critical threshold is reached when they will differentiate. The lineage of differentiation then becomes a sensitive function of both mechanics and morphology for a given chemical composition. Since PI is a major component of Gutta-percha, the FDA approved material commonly used for obturating the root canal, this work suggests that it can easily be adapted for in vivo use in dental regeneration.

ABA-type triblock copolymer micellar system with lower critical solution temperature-type sol-gel transition.

A temperature sensitive sol-gel transition induced by the self-assembly of amphiphilic copolymers and its application in industry have been the objects of increasing study. We demonstrate here a two-step, reversible addition-fragmentation chain transfer (RAFT) polymerization of an ABA-type copolymer consisting of poly(N,N-dimethylacrylamide)-b-poly(diacetone acrylamide)-b-poly(N,N-dimethylacrylamide) (PDMAA-b-PDAAM-b-PDMAA). This copolymer can be easily dispersed in water, and this dispersion is critical for its lower critical solution temperature (LCST)-type sol-gel transition, which was monitored using dynamic light scattering (DLS), transmission electron microscopy (TEM), and rheology analysis, in addition to temperature-dependent 1H nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR). Results revealed an abnormal sphere-to-worm micellar transition of this ABA copolymer at the LCST point, which could be affected by the length of the PDAAM block (B-block), the length as well as the distribution of the PDMAA block (A-block), and the concentration of the copolymer dispersion. Thus, copolymer dispersion could be feasibly used for drug loading at a low temperature, which could then be transformed into a gel at an elevated temperature. The loading and controllable release of the model drug of paracetamol into and out of a copolymer gel was further determined. The sustained release behavior was also studied using the Rigter-Peppas model.

Integrating optical coherence tomography with gravimetric and video analysis (OCT-Gravimetry-Video method) for studying the drying process of polystyrene latex system.

Latex, an aqueous dispersion of sub-micron polymer particles, is widely used as polymer binder in waterborne coatings and adhesives. Drying of a latex is inhomogeneous, during which the spatial distribution of particles is non-uniform and changes with time, usually resulting in a compromise of the integrity of a dried film. To study drying inhomogeneity of latex, we developed a system integrating optical coherence tomography (OCT) with gravimetric and video analysis (OCT-Gravimetry-Video method) to non-destructively monitor the drying process of non-film-forming latexes consisting of hard polystyrene spheres over time. OCT structural and speckle images of the latex's internal structure show the packing process of particles, the detachment of latex and the formation of apparent shear bands in cross-sectional views. Video recordings show the formation of cracks and the propagation of the drying boundary in the horizontal direction. The drying curve, measured by gravimetry, shows the drying rate and the water content of the latex at each drying stage. Furthermore, we find that the particle size affects packing and cracking phenomena remarkably. The OCT-Gravimetry-Video method serves as a general and robust approach to investigate the drying process of waterborne latex system. This method can be employed for fundamental studies of colloids and for evaluations of industrial latex products.

A novel dielectrophoresis potential spectroscopy for colloidal nanoparticles.

Dielectrophoresis (DEP) has been widely used to manipulate nanoparticles in microfluidic applications. However, determination of the DEP force of nanoparticles by theoretical models is not easy due to complications caused by the polarization of electrical double layer. Additionally, there is a lack of suitable experimental techniques to quantify the DEP force of nanoparticles. This article reports a statistical mechanics-based experimental method to determine the DEP potential energy of a single particle by measuring the equilibrium number density of particles in a DEP force field. Results show that at high frequencies, the measured potentials agree with the Maxwell-Wagner-O'Konski (MWO) theory. At frequencies lower than the crossover frequency (ωco ), the measured potential values are larger than MWO theory's predictions. When an effective particle radius (particle radius plus Debye length) is used to replace the particle radius, MWO theory fits better with the measured potentials on both sides of ωco . Also, the measured ωco was found inversely proportional to the effective particle radius, which agrees with MWO theory. The new DEP potential spectroscopy is not limited to the size or shape of particles, opening doors to investigate the DEP response functions of quantum dots and proteins in an alternating current electric field.

Lateral optical binding between two colloidal particles.

An optical binding force between two nearby colloidal particles trapped by two coherent laser beams is measured by phase-sensitive detection. The binding force is long-range and spatially oscillatory. For identical linearly-polarized incident beams, the oscillation period is equal to the optical wavelength. For mutually perpendicular polarizations, a new force appears with half-wavelength periodicity, caused by double inter-particle scattering. This force is observable only with cross-polarized incident beams, for which the stronger single-scattering forces are forbidden by parity.

Osmotic Bulk Modulus of Charged Colloids Measured by Ensemble Optical Trapping.

The optical-bottle technique is used to measure osmotic bulk moduli of colloid suspensions. The bulk modulus is determined by optically trapping an ensemble of nanoparticles and invoking a steady-state force balance between confining optical-gradient forces and repulsive osmotic-pressure forces. Osmotic bulk moduli are reported for aqueous suspensions of charged polystyrene particles in NaCl solutions as a function of particle concentration and ionic strength, and are compared to those determined by turbidity measurements under the same conditions. Effective particle charges are calculated from the bulk moduli and are found to increase as a function of ionic strength, consistent with previously reported results.

Exposure to TiO2 nanoparticles increases Staphylococcus aureus infection of HeLa cells.

BACKGROUND: Titanium dioxide (TiO2) is one of the most common nanoparticles found in industry ranging from food additives to energy generation. Approximately four million tons of TiO2 particles are produced worldwide each year with approximately 3000 tons being produced in nanoparticulate form, hence exposure to these particles is almost certain. RESULTS: Even though TiO2 is also used as an anti-bacterial agent in combination with UV, we have found that, in the absence of UV, exposure of HeLa cells to TiO2 nanoparticles significantly increased their risk of bacterial invasion. HeLa cells cultured with 0.1 mg/ml rutile and anatase TiO2 nanoparticles for 24 h prior to exposure to bacteria had 350 and 250 % respectively more bacteria per cell. The increase was attributed to bacterial polysaccharides absorption on TiO2 NPs, increased extracellular LDH, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO2 particles ingested 40 % fewer bacteria, further increasing the risk of infection. CONCLUSIONS: In combination, these two factors raise serious concerns regarding the impact of exposure to TiO2 nanoparticles on the ability of organisms to resist bacterial infection.

Low-frequency dielectrophoretic response of a single particle in aqueous suspensions.

We use optical tweezers-based dielectrophoresis (DEP) force spectroscopy to investigate the roles of the electrical double layer in the AC dielectric response of an individual colloidal particle in an aqueous medium. Specifically, we measure the DEP crossover frequency as a function of particles size, medium viscosity, and temperature. Experimental results were compared to low frequency relaxation mechanisms predicted by Schwarz, demonstrating the dielectrophoretic responses in the frequency range between 10 kHz and 1 MHz were dominated by counterion diffusion within the electric double layer.

Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization.

The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

Potential energy profile of colloidal nanoparticles in optical confinement.

An optical bottle method is developed to determine the potential-energy profile of colloidal Rayleigh nanoparticles in an optical trap. The three-dimensional distribution of fluorescent particles in the trap is measured by laser scanning confocal fluorescence microscopy. At sufficiently low concentrations at which interactions between the particles are negligible, the single-particle trapping potential-energy profile is determined from the equilibrium number-density profile by use of the Boltzmann distribution. Fluorescence imaging as well as calculations based on a discrete dipole approximation show that effects due to scattering forces are negligible for polystyrene particles of size less than 10% of the wavelength of the trapping laser, thus justifying the assumption of conservative forces in the equilibrium potential-energy determinations. The new optical bottle method measures the entire two-dimensional trapping-potential profile for an individual nanoparticle without the restriction that only one particle be contained in the trap, thus obviating the need for high laser power.

The influence of size, shape and vessel geometry on nanoparticle distribution.

Nanoparticles (NPs) are emerging as promising carrier platforms for targeted drug delivery and imaging probes. To evaluate the delivery efficiency, it is important to predict the distribution of NPs within blood vessels. NP size, shape and vessel geometry are believed to influence its biodistribution in circulation. Whereas, the effect of size on nanoparticle distribution has been extensively studied, little is known about the shape and vessel geometry effect. This paper describes a computational model for NP transport and distribution in a mimetic branched blood vessel using combined NP Brownian dynamics and continuum fluid mechanics approaches. The simulation results indicate that NPs with smaller size and rod shape have higher binding capabilities as a result of smaller drag force and larger contact area. The binding dynamics of rod-shaped NPs is found to be dependent on their initial contact points and orientations to the wall. Higher concentration of NPs is observed in the bifurcation area compared to the straight section of the branched vessel. Moreover, it is found that Péclet number plays an important role in determining the fraction of NPs deposited in the branched region and the straight section. Simulation results also indicate that NP binding decreases with increased shear rate. Dynamic NP re-distribution from low to high shear rates is observed due to the non-uniform shear stress distribution over the branched channel. This study would provide valuable information for NP distribution in a complex vascular network.

Mapping alternating current electroosmotic flow at the dielectrophoresis crossover frequency of a colloidal probe.

AC electroosmotic (ACEO) flow above the gap between coplanar electrodes is mapped by the measurement of Stokes forces on an optically trapped polystyrene colloidal particle. E²-dependent forces on the probe particle are selected by amplitude modulation (AM) of the ACEO electric field (E) and lock-in detection at twice the AM frequency. E²-dependent DEP of the probe is eliminated by driving the ACEO at the probe's DEP crossover frequency. The location-independent DEP crossover frequency is determined, in a separate experiment, as the limiting frequency of zero horizontal force as the probe is moved toward the midpoint between the electrodes. The ACEO velocity field, uncoupled from probe DEP effects, was mapped in the region 1-9 µm above a 28 µm gap between the electrodes. By use of variously sized probes, each at its DEP crossover frequency, the frequency dependence of the ACEO flow was determined at a point 3 µm above the electrode gap and 4 µm from an electrode tip. At this location the ACEO flow was maximal at ∼117 kHz for a low salt solution. This optical trapping method, by eliminating DEP forces on the probe, provides unambiguous mapping of the ACEO velocity field.

The properties of gold nanospheres studied with dark field optical trapping.

We demonstrate trapping and characterization of multiple gold nanospheres with a setup composed of dark field imaging and optical tweezers. The number of trapped nanospheres is quantified by the overall dark-field scattering intensity. The spectra of the scattering intensity show that there is no interparticle coupling among trapped nanospheres when the density of nanospheres in the trap is low enough (less than 10 particles), while the density of nanosphere increases the interparticle coupling of nanospheres becomes obvious. In addition, the trapping potential of a single gold nanosphere is obtained by trapping an ensemble of gold nanospheres.

Dielectrophoresis force spectroscopy for colloidal clusters.

Optical trapping-based force spectroscopy was used to measure the frequency-dependent DEP forces and DEP crossover frequencies of colloidal polymethyl methacrylate spheres and clusters. A single sphere or cluster, held by an optical tweezer, was positioned near the center of a pair of gold-film electrodes where alternating current elecroosmosis flow was negligible. Use of amplitude modulation and phase-sensitive lock-in detection for accurate measurement of the DEP force yielded new insight into dielectric relaxation mechanisms near the crossover frequencies. On one hand, the size dependence of the DEP force near the crossover frequencies indicates that the dominant polarization mechanism is a volume effect. On the other hand, the power-law dependence of the crossover frequency on the particle radius with an exponent of -2 indicates the dielectric relaxation is more likely because of ionic diffusion across the particle surface, suggesting the dominant polarization mechanism may be a surface polarization effect. Better theories are needed to explain the experiment. Nevertheless, the strong size dependence of the crossover frequencies suggests the use of DEP for size sorting of micron-sized particles.

Interface shear microrheometer with an optically driven oscillating probe particle.

We report the first experimental demonstration of an active interfacial shear microrheometer (ISMR) that uses a particle trapped by oscillating optical tweezers (OT) to probe the shear modulus G(s)(*)(ω) of a gas/liquid interface. The most significant advantages of the oscillating OT in a rheology study are: (1) very high sensitivity compared to other active microrheology methods and (2) the ability to measure both the real and imaginary components of the complex shear modulus without relying on the use of Kramers-Kronig relation, which can be problematic at low frequencies for most of the passive methods. We demonstrate the utilities of our ISMR in two case studies: (1) a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayer and (2) a composite of poly(styrene sulfonate) and dioctadecyldimethylammonium at the air/water interface in regimes where no other active instruments can explore.

Elastic response of binary hard-sphere fluids.

We derive expressions for the high-frequency, wave-number-dependent elastic constants of a binary hard-sphere fluid and employ Monte Carlo computer simulation to evaluate these constants in order to highlight the impact of composition and relative sphere diameter on the elastic response of this system. It is found that the elastic constant c(11)(k) exhibits oscillatory behavior as a function of k whereas the high-frequency shear modulus, for example, does not. This behavior is shown to be dictated by the angular dependence (in k[over ⃗] space) of derivatives of the interatomic force at contact. The results are related to recent measurements of the compressibility of colloidal fluids in laser trapping experiments.

Ensemble method to measure the potential energy of nanoparticles in an optical trap.

A method is described for measuring the potential energy of nanoparticles in an optical trap by trapping an ensemble of particles with a focused laser beam. The force balance between repulsive osmotic and confining gradient-force pressures determines the single-particle trapping potential independent of interactions between the particles. The ensemble nature of the measurement permits evaluation of single-particle trapping energies much smaller than kBT. Energies obtained by this method are compared to those of single-particle methods as well as to theoretical calculations based on classical electromagnetic optics.