Mechanical robustness of the cell under different modes of stress and deformation is essential to its survival and function. Under tension, mechanical rigidity is provided by the cytoskeletal network; with increasing stress, this network stiffens, providing increased resistance to deformation. However, a cell must also resist compression, which will inevitably occur whenever cell volume is decreased during such biologically important processes as anhydrobiosis and apoptosis. Under compression, individual filaments can buckle, thereby reducing the stiffness and weakening the cytoskeletal network. However, the intracellular space is crowded with macromolecules and organelles that can resist compression. A simple picture describing their behavior is that of colloidal particles; colloids exhibit a sharp increase in viscosity with increasing volume fraction, ultimately undergoing a glass transition and becoming a solid. We investigate the consequences of these 2 competing effects and show that as a cell is compressed by hyperosmotic stress it becomes progressively more rigid. Although this stiffening behavior depends somewhat on cell type, starting conditions, molecular motors, and cytoskeletal contributions, its dependence on solid volume fraction is exponential in every instance. This universal behavior suggests that compression-induced weakening of the network is overwhelmed by crowding-induced stiffening of the cytoplasm. We also show that compression dramatically slows intracellular relaxation processes. The increase in stiffness, combined with the slowing of relaxation processes, is reminiscent of a glass transition of colloidal suspensions, but only when comprised of deformable particles. Our work provides a means to probe the physical nature of the cytoplasm under compression, and leads to results that are universal across cell type.
Tanaka's approach to swelling kinetics of a solid gel sphere is extended to a spherical microgel shell. The boundary condition at the inner surface is obtained from the minimization of shear elastic energy. Temporal evolution of a shell is represented in a form of expansion over eigenfunctions of the corresponding diffusion equation. The swelling of Tanaka's solid spherical gel is recovered as a special case of our general Solution if the inner radius approaches zero. In another limiting case of it thin (balloon-like) shell, the set of eigenfunctions is reduced to a single exponential term. In the general case, a solid sphere swells slightly faster than the same sphere with in internal cavity. To test Our theoretical model, we prepared monodisperse poly-N-isopropylacrylamide (PNIPAM) hydrogel shells using a microfluidic device. The temporal dependence of the inner and outer radii of the shell were measured, and the data were fitted to our theoretical model. As a result, we obtained the collective diffusion constants for shrinking and for swelling processes. The obtained values for microgel shells are in excellent agreement with the previous results obtained for submillimeter PNIPAM solid spheres in the same temperature interval. Our model shows that the characteristic swelling time of a gel shell should be proportional to the square of the outer radius not to the thickness of the shell, agreeing with experimental observation.
Fundamental biological processes including morphogenesis, tissue repair and tumour metastasis require collective cell motions(1-3), and to drive these motions cells exert traction forces on their surroundings(4). Current understanding emphasizes that these traction forces arise mainly in 'leader cells' at the front edge of the advancing cell sheet(5-9). Our data are contrary to that assumption and show for the first time by direct measurement that traction forces driving collective cell migration arise predominately many cell rows behind the leading front edge and extend across enormous distances. Traction fluctuations are anomalous, moreover, exhibiting broad non-Gaussian distributions characterized by exponential tails(10-12). Taken together, these unexpected findings demonstrate that although the leader cell may have a pivotal role in local cell guidance, physical forces that it generates are but a small part of a global tug-of-war involving cells well back from the leading edge.
We describe a multi-color microfluidic dye laser operating in whispering gallery mode based on a train of alternating droplets containing solutions of different dyes; this laser is capable of switching the wavelength of its emission between 580 nm and 680 nm at frequencies up to 3.6 kHz-the fastest among all dye lasers reported; it has potential applications in on-chip spectroscopy and flow cytometry.
Colloidal and supracolloidal structures with anisotropic shape and surface chemistry are potential building blocks for the fabrication of novel materials. Droplets or bubbles are often used as templates for the assembly of particles into supracolloidal structures of spherical shape. Particle-coated droplets or bubbles have recently been shown to also retain nonspherical geometries after deformation, suggesting that the templating approach can also be used to produce supracolloidal structures with anisotropic shape. We show that partially coated droplets generated in a microcapillary device can undergo spontaneous coalescence into stable nonspherical structures. By positioning the droplets into regular arrays before coalescence, we produce anisotropic geometries with well-defined bonding angles between adjacent merged droplets. This approach allows for the fabrication of novel anisotropic supracolloidal structures with deliberately designed shapes.
We introduce a novel approach for synthesizing mesoporous hydroxyapatite (HAp, (Ca)(10), (PO(4))(6)(OH)(2)) using double emulsion droplets as microreactors. By using capillary microfluidic techniques, the size and the geometry of the droplet microreactors can be tuned easily. Double emulsion droplets offer the combined advantages of both shielding the reactants and on-demand addition of reactants; this makes them highly versatile microreactors. Such droplet microreactors also enable simple visualization of the HAp formation process as well as control over the porosity in the HAp that is synthesized. Powder formed with our technique demonstrates a remarkable microstructure, as well as significantly enhanced BET specific average surface area and nanoscale porosity. Our results present a novel synthesis approach for controlling the nanoscale porosity and the morphology of inorganic particles using double emulsion droplets.
Biphasic Janus particles with a precisely tunable internal morphology are fabricated using a novel, versatile, and robust technique. This technique can be used in conjunction with microfluidics to produce monodisperse particles, or can be combined with bulk emulsification techniques to produce large quantities of particles.
We present a simple microfluidic device that uses an array of well-defined chambers to immobilize thousands of femtoliter-to picoliter-scale aqueous drops suspended in inert carrier oil. This device enables timelapse studies of large numbers of individual drops, while simultaneously enabling subsequent drop recovery.
Cells within a genetically identical population exhibit phenotypic variation that in some cases can persist across multiple generations. However, information about the temporal variation and familial dependence of protein levels remains hidden when studying the population as an ensemble. To correlate phenotypes with the age and genealogy of single cells over time, we developed a microfluidic device that enables us to track multiple lineages in parallel by trapping single cells and constraining them to grow in lines for as many as 8 divisions. To illustrate the utility of this method, we investigate lineages of cells expressing one of 3 naturally regulated proteins, each with a different representative expression behavior. Within lineages deriving from single cells, we observe genealogically related clusters of cells with similar phenotype; cluster sizes vary markedly among the 3 proteins, suggesting that the time scale of phenotypic persistence is protein-specific. Growing lines of cells also allows us to dynamically track temporal fluctuations in protein levels at the same time as pedigree relationships among the cells as they divide in the chambers. We observe bursts in expression levels of the heat shock protein Hsp12-GFP that occur simultaneously in mother and daughter cells. In contrast, the ribosomal protein Rps8b-GFP shows relatively constant levels of expression over time. This method is an essential step toward understanding the time scales of phenotypic variation and correlations in phenotype among single cells within a population.
Cells and organisms are remarkably robust: they alter the variety and levels of expressed genes and proteins in response to environmental stimuli, including temperature, chemicals, and the stiffness of their surroundings. Ultimately changes in gene and protein expression can result in a distinct phenotypic state, which in some cases is maintained over multiple generations; the ability to pass on a particular phenotypic state to progeny cells is critical for differentiation. Moreover, epigenetic regulation of phenotype is also thought to provide an evolutionary advantage for a population of cells adapting to a fluctuating environment on faster timescales than the occurrence of genetic mutations. However, simple methods to study patterns of gene and protein expression on multi-generational timescales are sparse. Here we describe a technique to study lineages of single cells over multiple generations using a microfluidic device; this reveals patterns of expression where protein levels are correlated across multiple generations. Such quantitative information of protein expression in the context of pedigree remains hidden when studying the population as an ensemble.
We present a confocal microscopy study of 1.55 mu m monodisperse silica hard spheres as they sediment and crystallize at the bottom wall of a container. If the particles sediment onto a feature less flat wall, the two bottom layers crystallize simultaneously and layerwise growth follows. If the wall is replaced by a hexagonal template, only layerwise growth occurs. Our results complement earlier numerical simulations and experiments on other colloidal systems.
Optical microscopy and multi-particle tracking are used to study hydrodynamic interactions of monodisperse polymethylmethacrylate (PMMA) spheres at a decalin-water interface. The short-time self-diffusion coefficient measured at low surface coverage has the form D(S)(S)(n) = alpha D(0)(1 - beta n), where n is the area fraction Occupied by the particles, and Do is the Stokes-Einstein diffusion coefficient in the bulk suspension of PMMA spheres in decalin. The measured values of a are found to be in good agreement with the numerical calculation for the drag coefficient of interfacial particles. The measured values of P differ from that obtained for bulk suspensions, indicating that hydrodynamic interactions between the particles have interesting new features at the interface.
Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. Methodology/Principal Findings: We determined the structure of the 23(rd) Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D beta-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress. Conclusions/Significance: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.
Glass formation in colloidal suspensions has many of the hallmarks of glass formation in molecular materials(1-5). For hard-sphere colloids, which interact only as a result of excluded volume, phase behaviour is controlled by volume fraction, phi; an increase in phi drives the system towards its glassy state, analogously to a decrease in temperature, T, in molecular systems. When phi increases above phi* approximate to 0.53, the viscosity starts to increase significantly, and the system eventually moves out of equilibrium at the glass transition, phi(g) approximate to 0.58, where particle crowding greatly restricts structural relaxation(1-4). The large particle size makes it possible to study both structure and dynamics with light scattering(1) and imaging(3,4); colloidal suspensions have therefore provided considerable insight into the glass transition. However, hard-sphere colloidal suspensions do not exhibit the same diversity of behaviour as molecular glasses. This is highlighted by the wide variation in behaviour observed for the viscosity or structural relaxation time, tau(alpha), when the glassy state is approached in supercooled molecular liquids(5). This variation is characterized by the unifying concept of fragility(5), which has spurred the search for a 'universal' description of dynamic arrest in glass-forming liquids. For 'fragile' liquids, tau(alpha) is highly sensitive to changes in T, whereas non-fragile, or 'strong', liquids show a much lower T sensitivity. In contrast, hard-sphere colloidal suspensions are restricted to fragile behaviour, as determined by their phi dependence(1,6), ultimately limiting their utility in the study of the glass transition. Here we show that deformable colloidal particles, when studied through their concentration dependence at fixed temperature, do exhibit the same variation in fragility as that observed in the T dependence of molecular liquids at fixed volume. Their fragility is dictated by elastic properties on the scale of individual colloidal particles. Furthermore, we find an equivalent effect in molecular systems, where elasticity directly reflects fragility. Colloidal suspensions may thus provide new insight into glass formation in molecular systems.
We study the compression of depletion gels under the influence of a gravitational stress by monitoring the time evolution of the gel interface and the local volume fraction, phi, inside the gel. We find f is not constant throughout the gel. Instead, there is a volume fraction gradient that develops and grows along the gel height as the compression process proceeds. Our results are correctly described by a non-linear poroelastic model that explicitly incorporates the phi-dependence of the gravitational, elastic and viscous stresses acting on the gel.
Many liquid metals exposed to air develop an oxide film on their outer surface. This film is sufficiently solid-like to provide mechanical stability to small liquid metal droplets, yet weak enough to allow the droplets to be malleable. These properties are useful in both micro-electronics and microfluidics; however, little is known about how to characterize them. Here we probe the elastic, yielding, and relaxation properties of oxide-coated gallium and eutectic gallium indium using a rheometer equipped with a parallel-plate geometry. By using parallel plates of different size, we show that surface stresses dominate bulk stresses. These experiments also demonstrate that the apparent elastic properties of the oxide film are highly sensitive to its strain history. Moreover, the apparent elasticity is sensitive to the stresses stored in the oxide skin. We probe these stresses and their time-dependence, with both torque and normal force measurements. We also characterize the time-dependence of the elasticity by observing free vibrations of the rheometer. We rationalize the strain history and time-dependence in terms of oxidation and show that despite this dependence, reproducible elasticity measurements can be obtained due to the ability of shear to produce a state that is independent of the strain history. (C) 2009 The Society of Rheology. [DOI: 10.1122/1.3236517]