Encapsulated bacteria deform lipid vesicles into flagellated swimmers.

We study a synthetic system of motile Escherichia coli bacteria encapsulated inside giant lipid vesicles. Forces exerted by the bacteria on the inner side of the membrane are sufficient to extrude membrane tubes filled with one or several bacteria. We show that a physical coupling between the membrane tube and the flagella of the enclosed cells transforms the tube into an effective helical flagellum propelling the vesicle. We develop a simple theoretical model to estimate the propulsive force from the speed of the vesicles and demonstrate the good efficiency of this coupling mechanism. Together, these results point to design principles for conferring motility to synthetic cells.

Characterization and Control of the Run-and-Tumble Dynamics of Escherichia Coli.

We characterize the full spatiotemporal gait of populations of swimming Escherichia coli using renewal processes to analyze the measurements of intermediate scattering functions. This allows us to demonstrate quantitatively how the persistence length of an engineered strain can be controlled by a chemical inducer and to report a controlled transition from perpetual tumbling to smooth swimming. For wild-type E. coli, we measure simultaneously the microscopic motility parameters and the large-scale effective diffusivity, hence quantitatively bridging for the first time small-scale directed swimming and macroscopic diffusion.

Correction: Sizing multimodal suspensions with differential dynamic microscopy.

Correction for 'Sizing multimodal suspensions with differential dynamic microscopy' by Joe J. Bradley et al., Soft Matter, 2023, 19, 8179-8192, https://doi.org/10.1039/D3SM00593C.

Sizing multimodal suspensions with differential dynamic microscopy.

Differential dynamic microscopy (DDM) can be used to extract the mean particle size from videos of suspensions. However, many suspensions have multimodal particle size distributions, for which a single 'mean' is not a sufficient description. After clarifying how different particle sizes contribute to the signal in DDM, we show that standard DDM analysis can extract the mean sizes of two populations in a bimodal suspension given prior knowledge of the sample's bimodality. Further, the use of the CONTIN algorithm obviates the need for such prior knowledge. Finally, we show that by selectively analysing portions of the DDM images, we can size a trimodal suspension where the large particles would otherwise dominate the signal, again without prior knowledge of the trimodality.

A combined rheometry and imaging study of viscosity reduction in bacterial suspensions.

Suspending self-propelled "pushers" in a liquid lowers its viscosity. We study how this phenomenon depends on system size in bacterial suspensions using bulk rheometry and particle-tracking rheoimaging. Above the critical bacterial volume fraction needed to decrease the viscosity to zero, [Formula: see text], large-scale collective motion emerges in the quiescent state, and the flow becomes nonlinear. We confirm a theoretical prediction that such instability should be suppressed by confinement. Our results also show that a recent application of active liquid-crystal theory to such systems is untenable.

Run-to-Tumble Variability Controls the Surface Residence Times of E. coli Bacteria.

Motile bacteria are known to accumulate at surfaces, eventually leading to changes in bacterial motility and biofilm formation. We use a novel two-color, three-dimensional Lagrangian tracking technique to follow simultaneously the body and the flagella of a wild-type Escherichia coli. We observe long surface residence times and surface escape corresponding mostly to immediately antecedent tumbling. A motility model accounting for a large behavioral variability in run-time duration reproduces all experimental findings and gives new insights into surface trapping efficiency.

Probing the dynamics of turbid colloidal suspensions using differential dynamic microscopy.

Few techniques can reliably measure the dynamics of colloidal suspensions or other soft materials over a wide range of turbidities. Here we systematically investigate the capability of Differential Dynamic Microscopy (DDM) to characterise particle dynamics in turbid colloidal suspensions based on brightfield optical microscopy. We measure the Intermediate Scattering Function (ISF) of polystyrene microspheres suspended in water over a range of concentrations, turbidities, and up to 4 orders of magnitude in time-scales. These DDM results are compared to data obtained from both Dynamic Light Scattering (DLS) and Two-colour Dynamic Light Scattering (TCDLS). The latter allows for suppression of multiple scattering for moderately turbid suspensions. We find that DDM can obtain reliable diffusion coefficients at up to 10 and 1000 times higher particle concentrations than TCDLS and standard DLS, respectively. Additionally, we investigate the roles of the four length-scales relevant when imaging a suspension: the sample thickness L, the imaging depth z, the imaging depth of field DoF, and the photon mean free path . More detailed experiments and analysis reveal the appearance of a short-time process as turbidity is increased, which we associate with multiple scattering events within the imaging depth of the field. The long-time process corresponds to the particle dynamics from which particle-size can be estimated in the case of non-interacting particles. Finally, we provide a simple theoretical framework, ms-DDM, for turbid samples, which accounts for multiple scattering.

Hook length of the bacterial flagellum is optimized for maximal stability of the flagellar bundle.

Most bacteria swim in liquid environments by rotating one or several flagella. The long external filament of the flagellum is connected to a membrane-embedded basal body by a flexible universal joint, the hook, which allows the transmission of motor torque to the filament. The length of the hook is controlled on a nanometer scale by a sophisticated molecular ruler mechanism. However, why its length is stringently controlled has remained elusive. We engineered and studied a diverse set of hook-length variants of Salmonella enterica. Measurements of plate-assay motility, single-cell swimming speed, and directional persistence in quasi-2D and population-averaged swimming speed and body angular velocity in 3D revealed that the motility performance is optimal around the wild-type hook length. We conclude that too-short hooks may be too stiff to function as a junction and too-long hooks may buckle and create instability in the flagellar bundle. Accordingly, peritrichously flagellated bacteria move most efficiently as the distance travelled per body rotation is maximal and body wobbling is minimized. Thus, our results suggest that the molecular ruler mechanism evolved to control flagellar hook growth to the optimal length consistent with efficient bundle formation. The hook-length control mechanism is therefore a prime example of how bacteria evolved elegant but robust mechanisms to maximize their fitness under specific environmental constraints.

Characterising shear-induced dynamics in flowing complex fluids using differential dynamic microscopy.

Microscopic dynamics reveal the origin of the bulk rheological response in complex fluids. In model systems particle motion can be tracked, but for industrially relevant samples this is often impossible. Here we adapt differential dynamic microscopy (DDM) to study flowing highly-concentrated samples without particle resolution. By combining an investigation of oscillatory flow, using a novel "echo-DDM" analysis, and steady shear, through flow-DDM, we characterise the yielding of a silicone oil emulsion on both the microscopic and bulk level. Through measuring the rate of shear-induced droplet rearrangements and the flow velocity, the transition from a solid-like to liquid-like state is shown to occur in two steps: with droplet mobilisation marking the limit of linear visco-elasticity, followed by the development of shear localisation and macroscopic yielding. Using this suite of techniques, such insight could be developed for a wide variety of challenging complex fluids.

Particle sizing for flowing colloidal suspensions using flow-differential dynamic microscopy.

Particle size is a key variable in understanding the behaviour of the particulate products that underpin much of our modern lives. Typically obtained from suspensions at rest, measuring the particle size under flowing conditions would enable advances for in-line testing during manufacture and high-throughput testing during development. However, samples are often turbid, multiply scattering light and preventing the direct use of common sizing techniques. Differential dynamic microscopy (DDM) is a powerful technique for analysing video microscopy of such samples, measuring diffusion and hence particle size without the need to resolve individual particles while free of substantial user input. However, when applying DDM to a flowing sample, diffusive dynamics are rapidly dominated by flow effects, preventing particle sizing. Here, we develop "flow-DDM", a novel analysis scheme that combines optimised imaging conditions, a drift-velocity correction and modelling of the impact of flow. Flow-DDM allows a decoupling of flow from diffusive motion that facilitates successful particle size measurements at flow speeds an order of magnitude higher than for DDM. We demonstrate the generality of the technique by applying flow-DDM to two separate microscopy methods and flow geometries.

Osmotaxis in Escherichia coli through changes in motor speed.

Bacterial motility, and in particular repulsion or attraction toward specific chemicals, has been a subject of investigation for over 100 years, resulting in detailed understanding of bacterial chemotaxis and the corresponding sensory network in many bacterial species. For Escherichia coli most of the current understanding comes from the experiments with low levels of chemotactically active ligands. However, chemotactically inactive chemical species at concentrations found in the human gastrointestinal tract produce significant changes in E. coli's osmotic pressure and have been shown to lead to taxis. To understand how these nonspecific physical signals influence motility, we look at the response of individual bacterial flagellar motors under stepwise changes in external osmolarity. We combine these measurements with a population swimming assay under the same conditions. Unlike for chemotactic response, a long-term increase in swimming/motor speeds is observed, and in the motor rotational bias, both of which scale with the osmotic shock magnitude. We discuss how the speed changes we observe can lead to steady-state bacterial accumulation.

Coiled Coil Affinity-Based Systems for the Controlled Release of Biofunctionalized Gold Nanoparticles from Alginate Hydrogels.

Affinity-based systems represent a promising solution to control the delivery of therapeutics using hydrogels. Here, we report a hybrid system that is based on the peptidic E/K coiled coil affinity pair to mediate the release of gold nanoparticles (NPs) from alginate scaffolds. On one hand, the gold NPs were functionalized with the Ecoil-tagged epidermal growth factor (EGF). The bioactivity of the grafted EGF and the bioavailability of the Ecoil moiety were confirmed by EGF receptor phosphorylation assays and by capturing the functionalized NPs on a Kcoil-derivatized surface. On the other hand, alginate chains were modified with azido-homoalanine Kcoil (Aha-Kcoil) by azide-alkyne click chemistry. The hybrid system was formed by dispersing NPs functionalized with the Ecoil-tagged EGF in alginate hydrogels containing either 0, 10, or 20% of Kcoil-modified alginate (Alg-Kcoil). With 20% of Alg-Kcoil, the release of Ecoil-functionalized NPs was reduced by half when compared to the release of NPs without Ecoil, whereas little to no differences were noticed with either 0 or 10% of Alg-Kcoil. Differential dynamic microscopy was used to determine the diffusion coefficient of the NPs, and the results showed a decrease in the diffusion coefficient of Ecoil-functionalized NPs, when compared to bare PEGylated NPs. Altogether, our data demonstrated that the E/K coiled coil system can control the release of NPs in a high Kcoil content alginate gel, opening diverse applications in drug delivery.

Dynamic optical rectification and delivery of active particles.

We use moving light patterns to control the motion of Escherichia coli bacteria whose motility is photo-activated. Varying the pattern speed controls the magnitude and direction of the bacterial flux, and therefore the accumulation of cells in up- and down-stream reservoirs. We validate our results with two-dimensional simulations and a 1-dimensional analytic model, and use these to explore parameter space. We find that cell accumulation is controlled by a competition between directed flux and undirected, stochastic transport. Our results point to a number of design principles for using moving light patterns and light-activated micro-swimmers in a range of practical applications.

Probing the Spatiotemporal Dynamics of Catalytic Janus Particles with Single-Particle Tracking and Differential Dynamic Microscopy.

We demonstrate differential dynamic microscopy and particle tracking for the characterization of the spatiotemporal behavior of active Janus colloids in terms of the intermediate scattering function (ISF). We provide an analytical solution for the ISF of the paradigmatic active Brownian particle model and find striking agreement with experimental results from the smallest length scales, where translational diffusion and self-propulsion dominate, up to the largest ones, which probe effective diffusion due to rotational Brownian motion. At intermediate length scales, characteristic oscillations resolve the crossover between directed motion to orientational relaxation and allow us to discriminate active Brownian motion from other reorientation processes, e.g., run-and-tumble motion. A direct comparison to theoretical predictions reliably yields the rotational and translational diffusion coefficients of the particles, the mean and width of their speed distribution, and the temporal evolution of these parameters.

Flagellated bacterial motility in polymer solutions.

It is widely believed that the swimming speed, v, of many flagellated bacteria is a nonmonotonic function of the concentration, c, of high-molecular-weight linear polymers in aqueous solution, showing peaked v(c) curves. Pores in the polymer solution were suggested as the explanation. Quantifying this picture led to a theory that predicted peaked v(c) curves. Using high-throughput methods for characterizing motility, we measured v and the angular frequency of cell body rotation, Ω, of motile Escherichia coli as a function of polymer concentration in polyvinylpyrrolidone (PVP) and Ficoll solutions of different molecular weights. We find that nonmonotonic v(c) curves are typically due to low-molecular-weight impurities. After purification by dialysis, the measured v(c) and Ω(c) relations for all but the highest-molecular-weight PVP can be described in detail by Newtonian hydrodynamics. There is clear evidence for non-Newtonian effects in the highest-molecular-weight PVP solution. Calculations suggest that this is due to the fast-rotating flagella seeing a lower viscosity than the cell body, so that flagella can be seen as nano-rheometers for probing the non-Newtonian behavior of high polymer solutions on a molecular scale.

Quantitative characterization of run-and-tumble statistics in bulk bacterial suspensions.

We introduce a numerical method to extract the parameters of run-and-tumble dynamics from experimental measurements of the intermediate scattering function. We show that proceeding in Laplace space is unpractical and employ instead renewal processes to work directly in real time. We first validate our approach against data produced using agent-based simulations. This allows us to identify the length and time scales required for an accurate measurement of the motility parameters, including tumbling frequency and swim speed. We compare different models for the run-and-tumble dynamics by accounting for speed variability at the single-cell and population level, respectively. Finally, we apply our approach to experimental data on wild-type Escherichia coli obtained using differential dynamic microscopy.

Distinct types of multicellular aggregates in Pseudomonas aeruginosa liquid cultures.

Pseudomonas aeruginosa forms suspended multicellular aggregates when cultured in liquid media. These aggregates may be important in disease, and/or as a pathway to biofilm formation. The polysaccharide Psl and extracellular DNA (eDNA) have both been implicated in aggregation, but previous results depend strongly on the experimental conditions. Here we develop a quantitative microscopy-based method for assessing changes in the size distribution of suspended aggregates over time in growing cultures. For exponentially growing cultures of P. aeruginosa PAO1, we find that aggregation is mediated by cell-associated Psl, rather than by either eDNA or secreted Psl. These aggregates arise de novo within the culture via a growth process that involves both collisions and clonal growth, and Psl non-producing cells do not aggregate with producers. In contrast, we find that stationary phase (overnight) cultures contain a different type of multicellular aggregate, in which both eDNA and Psl mediate cohesion. Our findings suggest that the physical and biological properties of multicellular aggregates may be very different in early-stage vs late-stage bacterial cultures.

Differential dynamic microscopy: a high-throughput method for characterizing the motility of microorganisms.

We present a fast, high-throughput method for characterizing the motility of microorganisms in three dimensions based on standard imaging microscopy. Instead of tracking individual cells, we analyze the spatiotemporal fluctuations of the intensity in the sample from time-lapse images and obtain the intermediate scattering function of the system. We demonstrate our method on two different types of microorganisms: the bacterium Escherichia coli (both smooth swimming and wild type) and the biflagellate alga Chlamydomonas reinhardtii. We validate the methodology using computer simulations and particle tracking. From the intermediate scattering function, we are able to extract the swimming speed distribution, fraction of motile cells, and diffusivity for E. coli, and the swimming speed distribution, and amplitude and frequency of the oscillatory dynamics for C. reinhardtii. In both cases, the motility parameters were averaged over ∼10(4) cells and obtained in a few minutes.

Differential dynamic microscopy for anisotropic colloidal dynamics.

Differential dynamic microscopy (DDM) is a low-cost, high-throughput technique recently developed for characterizing the isotropic diffusion of spherical colloids using white-light optical microscopy. (1) We develop the theory for applying DDM to probe the dynamics of anisotropic colloidal samples such as various ordered phases, or particles interacting with an external field. The q-dependent dynamics can be measured in any direction in the image plane. We demonstrate the method on a dilute aqueous dispersion of anisotropic magnetic particles (hematite) aligned in a magnetic field. The measured diffusion coefficients parallel and perpendicular to the field direction are in good agreement with theoretical values. We show how these measurements allow us to extract the orientational order parameter S(2) of the system.

Scratching the Surface of the Protein Corona: Challenging Measurements and Controversies.

This Review aims to provide a systematic analysis of the literature regarding ongoing debates in protein corona research. Our goal is to portray the current understanding of two fundamental and debated characteristics of the protein corona, namely, the formation of mono- or multilayers of proteins and their binding (ir)reversibility. The statistical analysis we perform reveals that these characterisitics are strongly correlated to some physicochemical factors of the NP-protein system (particle size, bulk material, protein type), whereas the technique of investigation or the type of measurement (in situ or ex situ) do not impact the results, unlike commonly assumed. Regarding the binding reversibility, the experimental design (either dilution or competition experiments) is also shown to be a key factor, probably due to nontrivial protein binding mechanisms, which could explain the paradoxical phenomena reported in the literature. Overall, we suggest that to truly predict and control the protein corona, future efforts should be directed toward the mechanistic aspects of protein adsorption.