Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based Microfluidics.

Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor- and capital-intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop-based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5-fold catalytic efficiency.

Dynamic Speckle Holography.

We introduce dynamic speckle holography, a new technique that combines imaging and scattering to measure three-dimensional maps of displacements as small as ten nanometers over several centimeters, greatly extending the capabilities of traditional imaging systems. We attain this sensitivity by imaging speckle patterns of light collected at three scattering angles and measuring the decay in the temporal correlation due to local motion. We use dynamic speckle holography to measure the strain field of a colloidal gel undergoing fracture and establish the surprising role of internal tension in driving the fracture.

Self-Limited Accumulation of Colloids in Porous Media.

We present local direct imaging of the progressive adsorption of colloidal particles inside a 3D model porous medium. By varying the interparticle electrostatic interactions, we observe a large range of particle deposition regimes, from a single layer of particles at the surface of the medium to multiple layers and eventually clogging of the system. We derive the complete deposition dynamics and show that colloid accumulation is a self-limited mechanism towards a deposited fraction associated with a balance between the particle interactions and the imposed flow rate. These trends are explained and predicted using a simple probability model considering the particle adsorption energy and the variation of the drag energy with evolving porosity. This constitutes a direct validation of speculated particle transport mechanisms, and a further understanding of accumulation mechanisms.

Triple Junction at the Triple Point Resolved on the Individual Particle Level.

At the triple point of a repulsive screened Coulomb system, a fcc crystal, a bcc crystal, and a fluid phase coexist. At their intersection, these three phases form a liquid groove, the triple junction. Using confocal microscopy, we resolve the triple junction on a single-particle level in a model system of charged PMMA colloids in a nonpolar solvent. The groove is found to be extremely deep and the incommensurate solid-solid interface to be very broad. Thermal fluctuations hence appear to dominate the solid-solid interface. This indicates a very low interfacial energy. The fcc-bcc interfacial energy is quantitatively determined based on Young's equation and, indeed, it is only about 1.3 times higher than the fcc-fluid interfacial energy close to the triple point.

Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A.

Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders such as cardiac failure and pulmonary injury. The actin cytoskeleton's connectivity enables it to transmit forces rapidly over large distances, implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of ß-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with ß-integrin tails connecting to the actin cytoskeleton by binding directly to filamin. FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation. Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching, we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases ß-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.

Dynamic sound scattering: Field fluctuation spectroscopy with singly scattered ultrasound in the near and far fields.

Dynamic sound scattering (DSS) is a powerful acoustic technique for investigating the motion of particles or other inclusions inside an evolving medium. In DSS, this dynamic information is obtained by measuring the field autocorrelation function of the temporal fluctuations of singly scattered acoustic waves. The technique was initially introduced 15 years ago, but its technical aspects were not adequately discussed then. This paper addresses the need for a more complete account of the method by describing in detail two different implementations of this sound scattering technique, one of which is specifically adapted to a common experimental situation in ultrasonics. The technique is illustrated by the application of DSS to measure the mean square velocity fluctuations of particles in fluidized suspensions, as well as the dynamic velocity correlation length. By explaining the experimental and analytical methods involved in realizing the DSS technique in practice, the use of DSS will be facilitated for future studies of particulate suspension dynamics and particle properties over a wide range of particle sizes and concentrations, from millimeters down to nanometers, where the use of optical techniques is often limited by the opacity of the medium.

Spatial propagation of protein polymerization.

We consider the spatial dependence of filamentous protein self-assembly. Through studying the cases where the spreading of aggregated material is dominated either by diffusion or by growth, we derive analytical results for the spatial evolution of filamentous protein aggregation, which we validate against Monte Carlo simulations. Moreover, we compare the predictions of our theory with experimental measurements of two systems for which we identify the propagation as either growth or diffusion controlled. Our results connect the macroscopic observables that characterize the spatial propagation of protein self-assembly with the underlying microscopic processes and provide physical limits on spatial propagation and prionlike behavior associated with protein aggregation.

Propagation of extended fractures by local nucleation and rapid transverse expansion of crack-front distortion.

Fractures are ubiquitous and can lead to the catastrophic material failure of materials. Although fracturing in a two-dimensional plane is well understood, all fractures are extended in and propagate through three-dimensional space. Moreover, their behaviour is complex. Here we show that the forward propagation of a fracture front occurs through an initial rupture, nucleated at some localized position, followed by a very rapid transverse expansion at velocities as high as the Rayleigh-wave speed. We study fracturing in a circular geometry that achieves an uninterrupted extended fracture front and use a fluid to control the loading conditions that determine the amplitude of the forward jump. We find that this amplitude correlates with the transverse velocity. Dynamic rupture simulations capture the observations for only a high transverse velocity. These results highlight the importance of transverse dynamics in the forward propagation of an extended fracture.

Universal behavior of the osmotically compressed cell and its analogy to the colloidal glass transition.

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.

Optimal vein density in artificial and real leaves.

The long evolution of vascular plants has resulted in a tremendous variety of natural networks responsible for the evaporatively driven transport of water. Nevertheless, little is known about the physical principles that constrain vascular architecture. Inspired by plant leaves, we used microfluidic devices consisting of simple parallel channel networks in a polymeric material layer, permeable to water, to study the mechanisms of and the limits to evaporation-driven flow. We show that the flow rate through our biomimetic leaves increases linearly with channel density (1/d) until the distance between channels (d) is comparable with the thickness of the polymer layer (delta), above which the flow rate saturates. A comparison with the plant vascular networks shows that the same optimization criterion can be used to describe the placement of veins in leaves. These scaling relations for evaporatively driven flow through simple networks reveal basic design principles for the engineering of evaporation-permeation-driven devices, and highlight the role of physical constraints on the biological design of leaves.

The vortex-driven dynamics of droplets within droplets.

Understanding the fluid-structure interaction is crucial for an optimal design and manufacturing of soft mesoscale materials. Multi-core emulsions are a class of soft fluids assembled from cluster configurations of deformable oil-water double droplets (cores), often employed as building-blocks for the realisation of devices of interest in bio-technology, such as drug-delivery, tissue engineering and regenerative medicine. Here, we study the physics of multi-core emulsions flowing in microfluidic channels and report numerical evidence of a surprisingly rich variety of driven non-equilibrium states (NES), whose formation is caused by a dipolar fluid vortex triggered by the sheared structure of the flow carrier within the microchannel. The observed dynamic regimes range from long-lived NES at low core-area fraction, characterised by a planetary-like motion of the internal drops, to short-lived ones at high core-area fraction, in which a pre-chaotic motion results from multi-body collisions of inner drops, as combined with self-consistent hydrodynamic interactions. The onset of pre-chaotic behavior is marked by transitions of the cores from one vortex to another, a process that we interpret as manifestations of the system to maximize its entropy by filling voids, as they arise dynamically within the capsule.

Actin filament length tunes elasticity of flexibly cross-linked actin networks.

Networks of the cytoskeletal biopolymer actin cross-linked by the compliant protein filamin form soft gels that stiffen dramatically under shear stress. We demonstrate that the elasticity of these networks shows a strong dependence on the mean length of the actin polymers, unlike networks with small, rigid cross-links. This behavior is in agreement with a model of rigid filaments connected by multiple flexible linkers.

Surface acoustic wave actuated cell sorting (SAWACS).

We describe a novel microfluidic cell sorter which operates in continuous flow at high sorting rates. The device is based on a surface acoustic wave cell-sorting scheme and combines many advantages of fluorescence activated cell sorting (FACS) and fluorescence activated droplet sorting (FADS) in microfluidic channels. It is fully integrated on a PDMS device, and allows fast electronic control of cell diversion. We direct cells by acoustic streaming excited by a surface acoustic wave which deflects the fluid independently of the contrast in material properties of deflected objects and the continuous phase; thus the device underlying principle works without additional enhancement of the sorting by prior labelling of the cells with responsive markers such as magnetic or polarizable beads. Single cells are sorted directly from bulk media at rates as fast as several kHz without prior encapsulation into liquid droplet compartments as in traditional FACS. We have successfully directed HaCaT cells (human keratinocytes), fibroblasts from mice and MV3 melanoma cells. The low shear forces of this sorting method ensure that cells survive after sorting.

The role of polymer polydispersity in phase separation and gelation in colloid-polymer mixtures.

Mixtures of nonadsorbing polymer and colloidal particles exhibit a range of different morphologies depending on the particle and polymer concentrations and their relative size ratios. These can be very important for technological applications, where gelation can produce a weak solidlike structure that can help reduce phase separation, extending product shelf life. However, industrial products are typically formulated with polydisperse polymers, and the consequences of this on the phase behavior of the mixture are not known. We investigate the role of polymer polydispersity and show that a small amount of larger polymer in a distribution of nominally much smaller polymer can drastically modify the behavior. It can induce formation of a solidlike gel structure, abetted by the small polymer, but still allow further evolution of the phase separation process, as is seen with a monodisperse distribution of larger polymer. This coarsening ultimately leads to gravitational collapse. We describe the full phase behavior for polydisperse polymer mixtures and account for the origin of the behavior through measurements of the structure and dynamics and by comparing to the behavior with monodisperse polymers.

Filamin A is essential for active cell stiffening but not passive stiffening under external force.

The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. Some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical environment or biochemical cues. Here we show that the actin-binding protein filamin A is essential for the active stiffening of cells plated on collagen-coated substrates. This appears to be due to a diminished capability to build up large internal contractile stresses in the absence of filamin A. To show this, we compare the material properties and contractility of two human melanoma cell lines that differ in filamin A expression. The filamin A-deficient M2 cells are softer than the filamin A-replete A7 cells, and exert much smaller contractile stresses on the substratum, even though the M2 cells have similar levels of phosphorylated myosin II light chain and only somewhat diminished adhesion strength. In contrast to A7 cells, the stiffness and contractility of M2 cells are insensitive to either myosin-inhibiting drugs or the stiffness of the substratum. Surprisingly, however, filamin A is not required for passive stiffening under large external forces.

Multiple light-scattering probes of foam structure and dynamics.

The structure and dynamics of three-dimensional foams are probed quantitatively by exploiting the strong multiple scattering of light that gives foams their familiar white color. Approximating the propagation of light as a diffusion process, transmission measurements provide a direct probe of the average bubble size. A model for dynamic light scattering is developed that can be used to interpret temporal fluctuations in the intensity of multiply scattered light. The results identify previously unrecognized internal dynamics of the foam bubbles. These light-scattering techniques are direct, noninvasive probes of bulk foams and therefore should find wide use in the study of their properties.

Real-space imaging of nucleation and growth in colloidal crystallization.

Crystallization of concentrated colloidal suspensions was studied in real space with laser scanning confocal microscopy. Direct imaging in three dimensions allowed identification and observation of both nucleation and growth of crystalline regions, providing an experimental measure of properties of the nucleating crystallites. By following their evolution, we identified critical nuclei, determined nucleation rates, and measured the average surface tension of the crystal-liquid interface. The structure of the nuclei was the same as the bulk solid phase, random hexagonal close-packed, and their average shape was rather nonspherical, with rough rather than faceted surfaces.

Gravitational compression of colloidal gels.

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, φ, inside the gel. We find φ 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 φ-dependence of the gravitational, elastic and viscous stresses acting on the gel.

Experimental observation of the crystallization of hard-sphere colloidal particles by sedimentation onto flat and patterned surfaces.

We present a confocal microscopy study of 1.55 microm 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.

Impact of inlet channel geometry on microfluidic drop formation.

We study the impact of inlet channel geometry on microfluidic drop formation. We show that drop makers with T-junction style inlets form monodisperse emulsions at low and moderate capillary numbers and those with Flow-Focus style inlets do so at moderate and high capillary numbers. At low and moderate capillary number, drop formation is dominated by interfacial forces and mediated by the confinement of the microchannels; drop size as a function of flow-rate ratio follows a simple functional form based on a blocking-squeezing mechanism. We summarize the stability of the drop makers with different inlet channel geometry in the form of a phase diagram as a function of capillary number and flow-rate ratio.