Long-Term Stability, Biocompatibility, and Magnetization of Suspensions of Isolated Bacterial Magnetosomes.

Magnetosomes are magnetic nanoparticles biosynthesized by magnetotactic bacteria. Due to a genetically strictly controlled biomineralization process, the ensuing magnetosomes have been envisioned as agents for biomedical and clinical applications. In the present work, different stability parameters of magnetosomes isolated from Magnetospirillum gryphiswaldense upon storage in suspension (HEPES buffer, 4 °C, nitrogen atmosphere) for one year in the absence of antibiotics are examined. The magnetic potency, measured by the saturation magnetization of the particle suspension, drops to one-third of its starting value within this year-about ten times slower than at ambient air and room temperature. The particle size distribution, the integrity of the surrounding magnetosome membrane, the colloidal stability, and the biocompatibility turn out to be not severely affected by long-term storage.

Light-regulated adsorption and desorption of Chlamydomonas cells at surfaces.

Microbial colonization of surfaces represents the first step towards biofilm formation, which is a recurring phenomenon in nature with beneficial and detrimental implications in technological and medical settings. Consequently, there is interest in elucidating the fundamental aspects of the initial stages of biofilm formation of microorganisms on solid surfaces. While most of the research is oriented to understand bacterial surface colonization, the fundamental principles of surface colonization of motile, photosynthetic microbes remain largely unexplored so far. Recent single-cell studies showed that the flagellar adhesion of Chlamydomonas reinhardtii is switched on in blue light and switched off under red light [Kreis et al., Nat. Phys., 2018, 14, 45-49]. Here, we study this light-switchable surface association on the population level and measure the kinetics of adsorption and desorption of suspensions of motile C. reinhardtii cells on glass surfaces using bright-field optical microscopy. We observe that both processes exhibit a response lag relative to the time at which the blue- and red-light conditions are set and model this feature using time-delayed Langmuir-type kinetics. We find that cell adsorption occurs significantly faster than desorption, which we attribute to the protein-mediated molecular adhesion mechanism of the cells. Adsorption experiments using phototactically blind C. reinhardtii mutants demonstrate that phototaxis does not affect the cell adsorption kinetics. Hence, this framework can be used as an assay for characterizing the dynamics of the surface colonization of microbial species exhibiting light-regulated surface adhesion under precisely controlled environmental conditions.

Self-generated oxygen gradients control collective aggregation of photosynthetic microbes.

For billions of years, photosynthetic microbes have evolved under the variable exposure to sunlight in diverse ecosystems and microhabitats all over our planet. Their abilities to dynamically respond to alterations of the luminous intensity, including phototaxis, surface association and diurnal cell cycles, are pivotal for their survival. If these strategies fail in the absence of light, the microbes can still sustain essential metabolic functionalities and motility by switching their energy production from photosynthesis to oxygen respiration. For suspensions of motile C. reinhardtii cells above a critical density, we demonstrate that this switch reversibly controls collective microbial aggregation. Aerobic respiration dominates over photosynthesis in conditions of low light, which causes the microbial motility to sensitively depend on the local availability of oxygen. For dense microbial populations in self-generated oxygen gradients, microfluidic experiments and continuum theory based on a reaction-diffusion mechanism show that oxygen-regulated motility enables the collective emergence of highly localized regions of high and low cell densities.

Emergent probability fluxes in confined microbial navigation.

When the motion of a motile cell is observed closely, it appears erratic, and yet the combination of nonequilibrium forces and surfaces can produce striking examples of organization in microbial systems. While most of our current understanding is based on bulk systems or idealized geometries, it remains elusive how and at which length scale self-organization emerges in complex geometries. Here, using experiments and analytical and numerical calculations, we study the motion of motile cells under controlled microfluidic conditions and demonstrate that probability flux loops organize active motion, even at the level of a single cell exploring an isolated compartment of nontrivial geometry. By accounting for the interplay of activity and interfacial forces, we find that the boundary's curvature determines the nonequilibrium probability fluxes of the motion. We theoretically predict a universal relation between fluxes and global geometric properties that is directly confirmed by experiments. Our findings open the possibility to decipher the most probable trajectories of motile cells and may enable the design of geometries guiding their time-averaged motion.

Measuring and upscaling micromechanical interactions in a cohesive granular material.

The mechanical properties of a disordered heterogeneous medium depend, in general, on a complex interplay between multiple length scales. Connecting local interactions to macroscopic observables, such as stiffness or fracture, is thus challenging in this type of material. Here, we study the properties of a cohesive granular material composed of glass beads held together by soft polymer bridges. We characterise the mechanical response of single bridges under traction and shear, using a setup based on the deflection of flexible micropipettes. These measurements, along with information from X-ray microtomograms of the granular packings, then inform large-scale discrete element model (DEM) simulations. Although simple, these simulations are constrained in every way by empirical measurement and accurately predict mechanical responses of the aggregates, including details on their compressive failure, and how the material's stiffness depends on the stiffness and geometry of its parts. By demonstrating how to accurately relate microscopic information to macroscopic properties, these results provide new perspectives for predicting the behaviour of complex disordered materials, such as porous rock, snow, or foam.

Surfactant-free production of biomimetic giant unilamellar vesicles using PDMS-based microfluidics.

Microfluidic production of giant lipid vesicles presents a paradigm-shift in the development of artificial cells. While production is high-throughput and the lipid vesicles are mono-disperse compared to bulk methods, current technologies rely heavily on the addition of additives such as surfactants, glycerol and even ethanol. Here we present a microfluidic method for producing biomimetic surfactant-free and additive-free giant unilamellar vesicles. The versatile design allows for the production of vesicle sizes ranging anywhere from ~10 to 130 µm with either neutral or charged lipids, and in physiological buffer conditions. Purity, functionality, and stability of the membranes are validated by lipid diffusion, protein incorporation, and leakage assays. Usability as artificial cells is demonstrated by increasing their complexity, i.e., by encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres. This robust method capable of creating truly biomimetic artificial cells in high-throughput will prove valuable for bottom-up synthetic biology and the understanding of membrane function.

Altered N-glycan composition impacts flagella-mediated adhesion in Chlamydomonas reinhardtii.

For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. However, it is not known whether only the presence or also the composition of N-glycans attached to respective proteins is important for these processes. To this end, we tested several C. reinhardtii insertional mutants and a CRISPR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces. This results in impaired polystyrene bead binding and transport but not gliding of cells on solid surfaces. Notably, assembly, intraflagellar transport, and protein import into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact.

Dynamic force measurements on swimming Chlamydomonas cells using micropipette force sensors.

Flagella and cilia are cellular appendages that inherit essential functions of microbial life including sensing and navigating the environment. In order to propel a swimming microorganism they displace the surrounding fluid by means of periodic motions, while precisely timed modulations of their beating patterns enable the cell to steer towards or away from specific locations. Characterizing the dynamic forces, however, is challenging and typically relies on indirect experimental approaches. Here, we present direct in vivo measurements of the dynamic forces of motile Chlamydomonas reinhardtii cells in controlled environments. The experiments are based on partially aspirating a living microorganism at the tip of a micropipette force sensor and optically recording the micropipette's position fluctuations with high temporal and sub-pixel spatial resolution. Spectral signal analysis allows for isolating the cell-generated dynamic forces caused by the periodic motion of the flagella from background noise. We provide an analytic, elasto-hydrodynamic model for the micropipette force sensor and describe how to obtain the micropipette's full frequency response function from a dynamic force calibration. Using this approach, we measure the amplitude of the oscillatory forces during the swimming activity of individual Chlamydomonas reinhardtii cells of 26 ± 5 pN, resulting from the coordinated flagellar beating with a frequency of 49 ± 5 Hz. This dynamic micropipette force sensor technique generalizes the applicability of micropipettes as force sensors from static to dynamic force measurements, yielding a force sensitivity in the piconewton range. In addition to measurements in bulk liquid environment, we study the dynamic forces of the biflagellated microswimmer in the vicinity of a solid/liquid interface. As we gradually decrease the distance of the swimming microbe to the interface, we measure a significantly enhanced force transduction at distances larger than the maximum extent of the beating flagella, highlighting the importance of hydrodynamic interactions for scenarios in which flagellated microorganisms encounter surfaces.

In vivo adhesion force measurements of Chlamydomonas on model substrates.

The initial stages of biofilm formation at a surface are triggered by the surface association of individual microorganisms. The biological mechanisms and interfacial interactions underlying microbial adhesion to surfaces have been widely studied for bacteria, while microalgae remained rather unconsidered despite their technological relevance, e.g., in photo-bioreactors. We performed in vivo micropipette force measurements with the model organism Chlamydomonas reinhardtii, a unicellular eukaryotic microalga that dwells in liquid-infused soils and on moist rocks. We characterize the adhesion forces and dissect the influence of intermolecular interactions by probing the adhesion forces of single cells on different model substrates with tailored properties. Our experiments show that the flagella-mediated adhesion of Chlamydomonas to surfaces is largely substrate independent, enabling the cell to adhere to any type of surface. This universal adhesion mechanism allows the microalga to effectively colonize abiotic surfaces in their heterogeneous natural habitats. Our results reveal a dominant contribution of electrostatic interactions governing microalgal adhesion and suggest that flagella membrane processes may cause significant variations of the adhesive properties of the flagella.

Micropipette force sensors for in vivo force measurements on single cells and multicellular microorganisms.

Measuring forces from the piconewton to millinewton range is of great importance for the study of living systems from a biophysical perspective. The use of flexible micropipettes as highly sensitive force probes has become established in the biophysical community, advancing our understanding of cellular processes and microbial behavior. The micropipette force sensor (MFS) technique relies on measurement of the forces acting on a force-calibrated, hollow glass micropipette by optically detecting its deflections. The MFS technique covers a wide micro- and mesoscopic regime of detectable forces (tens of piconewtons to millinewtons) and sample sizes (micrometers to millimeters), does not require gluing of the sample to the cantilever, and allows simultaneous optical imaging of the sample throughout the experiment. Here, we provide a detailed protocol describing how to manufacture and calibrate the micropipettes, as well as how to successfully design, perform, and troubleshoot MFS experiments. We exemplify our approach using the model nematode Caenorhabditis elegans, but by following this protocol, a wide variety of living samples, ranging from single cells to multicellular aggregates and millimeter-sized organisms, can be studied in vivo, with a force resolution as low as 10 pN. A skilled (under)graduate student can master the technique in ~1-2 months. The whole protocol takes ~1-2 d to finish.

Adhesion strategies of Dictyostelium discoideum- a force spectroscopy study.

Biological adhesion is essential for all motile cells and generally limits locomotion to suitably functionalized substrates displaying a compatible surface chemistry. However, organisms that face vastly varying environmental challenges require a different strategy. The model organism Dictyostelium discoideum (D.d.), a slime mould dwelling in the soil, faces the challenge of overcoming variable chemistry by employing the fundamental forces of colloid science. To understand the origin of D.d. adhesion, we realized and modified a variety of conditions for the amoeba comprising the absence and presence of the specific adhesion protein Substrate Adhesion A (sadA), glycolytic degradation, ionic strength, surface hydrophobicity and strength of van der Waals interactions by generating tailored model substrates. By employing AFM-based single cell force spectroscopy we could show that experimental force curves upon retraction exhibit two regimes. The first part up to the critical adhesion force can be described in terms of a continuum model, while the second regime of the curve beyond the critical adhesion force is governed by stochastic unbinding of individual binding partners and bond clusters. We found that D.d. relies on adhesive interactions based on EDL-DLVO (Electrical Double Layer-Derjaguin-Landau-Verwey-Overbeek) forces and contributions from the glycocalix and specialized adhesion molecules like sadA. This versatile mechanism allows the cells to adhere to a large variety of natural surfaces under various conditions.

Adsorption-induced slip inhibition for polymer melts on ideal substrates.

Hydrodynamic slip, the motion of a liquid along a solid surface, represents a fundamental phenomenon in fluid dynamics that governs liquid transport at small scales. For polymeric liquids, de Gennes predicted that the Navier boundary condition together with polymer reptation implies extraordinarily large interfacial slip for entangled polymer melts on ideal surfaces; this Navier-de Gennes model was confirmed using dewetting experiments on ultra-smooth, low-energy substrates. Here, we use capillary leveling-surface tension driven flow of films with initially non-uniform thickness-of polymeric films on these same substrates. Measurement of the slip length from a robust one parameter fit to a lubrication model is achieved. We show that at the low shear rates involved in leveling experiments as compared to dewetting ones, the employed substrates can no longer be considered ideal. The data is instead consistent with a model that includes physical adsorption of polymer chains at the solid/liquid interface.

Curvature-Guided Motility of Microalgae in Geometric Confinement.

Microorganisms, such as bacteria and microalgae, often live in habitats consisting of a liquid phase and a plethora of interfaces. The precise ways in which these motile microbes behave in their confined environment remain unclear. Using experiments and Brownian dynamics simulations, we study the motility of a single Chlamydomonas microalga in an isolated microhabitat with controlled geometric properties. We demonstrate how the geometry of the habitat controls the cell's navigation in confinement. The probability of finding the cell swimming near the boundary increases with the wall curvature, as seen for both circular and elliptical chambers. The theory, utilizing an asymmetric dumbbell model of the cell and steric wall interactions, captures this curvature-guided navigation quantitatively with no free parameters.

A modular approach for multifunctional polymersomes with controlled adhesive properties.

The bottom-up approach in synthetic biology involves the engineering of synthetic cells by designing biological and chemical building blocks, which can be combined in order to mimic cellular functions. The first step for mimicking a living cell is the design of an appropriate compartment featuring a multifunctional membrane. This is of particular interest since it allows for the selective attachment of different groups or molecules to the membrane. In this context, we report on a modular approach for polymeric vesicles, so-called polymersomes, with a multifunctional surface, namely hydroxyl, alkyne and acrylate groups. We demonstrate that the surface of the polymersome can be functionalized to facilitate imaging, via fluorescent dyes, or to improve the specific adhesion to surfaces by using a biotin functionalization. This generally applicable multifunctionality allows for the covalent integration of various molecules in the membrane of a synthetic cell.

Nucleated dewetting in supported ultra-thin liquid films with hydrodynamic slip.

This study reveals the influence of the surface energy and solid/liquid boundary condition on the breakup mechanism of dewetting ultra-thin polymer films. Using silane self-assembled monolayers, SiO2 substrates are rendered hydrophobic and provide a strong slip rather than a no-slip solid/liquid boundary condition. On undergoing these changes, the thin-film breakup morphology changes dramatically - from a spinodal mechanism to a breakup which is governed by nucleation and growth. The experiments reveal a dependence of the hole density on film thickness and temperature. The combination of lowered surface energy and hydrodynamic slip brings the studied system closer to the conditions encountered in bursting unsupported films. As for unsupported polymer films, a critical nucleus size is inferred from a free energy model. This critical nucleus size is supported by the film breakup observed in the experiments using high speed in situ atomic force microscopy.

Vesicles-on-a-chip: A universal microfluidic platform for the assembly of liposomes and polymersomes.

In this study, we present a PDMS-based microfluidic platform for the fabrication of both liposomes and polymersomes. Based on a double-emulsion template formed in flow-focusing configuration, monodisperse liposomes and polymersomes are produced in a controlled manner after solvent extraction. Both types of vesicles can be formed from the exact same combination of fluids and are stable for at least three months under ambient storage conditions. By tuning the flow rates of the different fluid phases in the flow-focusing microfluidic design, the size of the liposomes and polymersomes can be varied over at least one order of magnitude. This method offers a versatile tool for future studies, e.g., involving the encapsulation of biological agents and the functionalization of artificial cell membranes, and might also be applicable for the controlled fabrication of hybrid vesicles.

Solid capillarity: when and how does surface tension deform soft solids?

Soft solids differ from stiff solids in an important way: their surface stresses can drive large deformations. Based on a topical workshop held in the Lorentz Center in Leiden, this Opinion highlights some recent advances in the growing field of solid capillarity and poses key questions for its advancement.

Universal contact-line dynamics at the nanoscale.

The relaxation dynamics of the contact angle between a viscous liquid and a smooth substrate is studied at the nanoscale. Through atomic force microscopy measurements of polystyrene nanostripes we simultaneously monitor both the temporal evolution of the liquid-air interface and the position of the contact line. The initial configuration exhibits high curvature gradients and a non-equilibrium contact angle that drive liquid flow. Both these conditions are relaxed to achieve the final state, leading to three successive regimes in time: (i) stationary contact line levelling; (ii) receding contact line dewetting; (iii) collapse of the two fronts. For the first regime, we reveal the existence of a self-similar evolution of the liquid interface, which is in excellent agreement with numerical calculations from a lubrication model. For different liquid viscosities and film thicknesses we provide evidence for a transition to dewetting featuring a universal critical contact angle and dimensionless time.

Onset of Area-Dependent Dissipation in Droplet Spreading.

We probe the viscous relaxation of structured liquid droplets in the partial wetting regime using a diblock copolymer system. The relaxation time of the droplets is measured after a step change in temperature as a function of three tunable parameters: droplet size, equilibrium contact angle, and the viscosity of the fluid. Contrary to what is typically observed, the late-stage relaxation time does not scale with the radius of the droplet-rather, relaxation scales with the radius squared. Thus, the energy dissipation depends on the contact area of the droplet, rather than the contact line.

Capillary droplet propulsion on a fibre.

A viscous liquid film coating a fibre becomes unstable and decays into droplets due to the Rayleigh-Plateau instability (RPI). Here, we report on the generation of uniform droplets on a hydrophobized fibre by taking advantage of this effect. In the late stages of liquid column breakup, a three-phase contact line can be formed at one side of the droplet by spontaneous rupture of the thinning film. The resulting capillary imbalance leads to droplet propulsion along the fibre. We study the dynamics and the dewetting speed of the droplet as a function of molecular weight as well as temperature and compare to a force balance model based on purely viscous dissipation.