Understanding the locomotion of microorganisms is essential for insights into microbial ecology, infection, and colonization processes. Although two-dimensional microscopy has been widely used to study microswimmer motility, it does not capture the full extent of their three-dimensional (3D) movement. Recent advances in defocused particle tracking, holographic tracking velocimetry, and stereo-microscopy face challenges in achieving high resolution at larger particle densities and tracking multiple microswimmers in suspension. In this work, we introduce a novel multi-camera microscopy system that significantly improves the accuracy of 3D microswimmer tracking. Our system uses four sCMOS cameras to image microorganisms within a 2.5 $$\times$$ × 2.5 $$\times$$ × 2 mm $$^3$$ 3 . We assess the performance of our microscopy system by tracking a population of the unicellular motile algae Chlamydomonas reinhardtii . An in-house tracking algorithm based on the projective geometry framework enables tracking with reprojection errors below 0.3 body lengths. This system supports imaging and tracking particle source densities of 0.32, higher than other existing single camera 3D microscopy techniques. Analysis of C. reinhardtii trajectories in 3D reveals a predominance of left-handed chirality and helical swimming patterns. Moreover, our 3D tracking data provide translational and rotational diffusion coefficients that differ from those obtained using traditional two-dimensional methods.

This paper presents a microfluidic approach that dynamically controls the hydrodynamic flow and the streamlines to enable complex multi-particle manipulations within a single device. The approach combines the design of a flow-through microfluidic Hele–Shaw flow cell together with an optimization procedure to find a priori optimal particle pathlines, and an effective proportional–integral–derivative (PID) feedback controller to provide real-time control over the particle manipulations. In the device, particles are manipulated with hydrodynamic forces, by using a uniform flow through the flow cell and three inlets perpendicular to the flow cell. The streamlines within the device are manipulated by injecting or extracting fluid through the three inlets. The Hele–Shaw geometry allows a fast and accurate prediction of the particle trajectory, meaning only a simple PID controller is required to correct for particle deviations. The robustness of this approach is demonstrated by implementing multiple functions within the device, including particle trapping, particle sorting, particle separation, and assembly. The real-time control procedure affords accurate particle manipulation, with a maximum error on the order of the diameter of the particle.

Eukaryotes swim with coordinated flagellar (ciliary) beating and steer by fine-tuning the coordination. The model organism for studying flagellate motility, Chlamydomonas reinhardtii, employs synchronous, breaststroke-like flagellar beating to swim, and it modulates the beating amplitudes differentially to steer. This strategy hinges on both inherent flagellar asymmetries (e.g. different response to chemical messengers) and such asymmetries being effectively coordinated in the synchronous beating. In C. reinhardtii, the synchrony of beating is known to be supported by a mechanical connection between flagella; however, how flagellar asymmetries persist in the synchrony remains elusive. For example, it has been speculated for decades that one flagellum leads the beating, as its dynamic properties (i.e. frequency, waveform, etc.) appear to be copied by the other one. In this study, we combine experiments, computations, and modeling efforts to elucidate the roles played by each flagellum in synchronous beating. With a non-invasive technique to selectively load each flagellum, we show that the coordinated beating essentially only responds to load exerted on the cis flagellum; and that such asymmetry in response derives from a unilateral coupling between the two flagella. Our results highlight a distinct role for each flagellum in coordination and have implication for biflagellates’ tactic behaviors.

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE), which make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating >30 mu m-sized droplets and only in some cases GUV-sized (similar to 15 mu m) satellite droplets. According to existing theoretical models, the oil-water interface should allow for the crossing of all droplets, but based on our observations and scaling arguments on the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. The origin of these droplets remains partly unclear; we hypothesize that some small GUVs might be formed from large droplets sitting at the second interface. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering the lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE but ultimately also aid in the development of more reliable and efficient methods for GUV production.

Coexistence of lipid domains in cell membranes is associated with vital biological processes. Here, we investigate two such membranes: a multi-component membrane composed of DOPC and DPPC lipids with gel and fluid separated domains, and a single component membrane composed of PMPC lipids forming ripples. We characterize their mechanical properties below their melting point, where ordered and disordered regions coexist, and above their melting point, where they are in fluid phase. To conduct these inquiries, we create lipid bilayers in a microfluidic chip interfaced with a heating system and optical tweezers. The chip features a bubble trap and enables high-throughput formation of planar bilayers. Optical tweezers experiments reveal interfacial hydrodynamics (fluid-slip) and elastic properties (membrane tension and bending rigidity) at various temperatures. For PMPC bilayers, we demonstrate a higher fluid slip at the interface in the fluid-phase compared to the ripple phase, while for the DOPC:DPPC mixture, similar fluid slip is measured below and above the transition point. Membrane tension for both compositions increases after thermal fluidization. Bending rigidity is also measured using the forces required to extend a lipid nanotube pushed out of the freestanding membranes. This novel temperature-controlled microfluidic platform opens numerous possibilities for thermomechanical studies on freestanding planar membranes. Lipid membranes formed in a microfluidic device are studied with temperature-controlled optical tweezers, revealing how mechanical properties of ripple phase and gel-fluid phase-separated membranes change when heated above the transition temperature.

Eukaryotes swim with coordinated flagellar (ciliary) beating and steer by fine-tuning the coordination. The model organism for studying flagellate motility, C. reinhardtii (CR), employs synchronous, breast-stroke-like flagellar beating to swim, and it modulates the beating amplitudes differentially to steer. This strategy hinges on both inherent flagellar asymmetries (e.g. different response to chemical messengers) and such asymmetries being effectively coordinated in the synchronous beating. In CR, the synchrony of beating is known to be supported by a mechanical connection between flagella, however, how flagellar asymmetries persist in the synchrony remains elusive. For example, it has been speculated for decades that one flagellum leads the beating, as its dynamic properties (i.e. frequency, waveform, etc.) appear to be copied by the other one. In this study, we combine experiments, computations, and modeling efforts to elucidate the roles played by each flagellum in synchronous beating. With a non-invasive technique to selectively load each flagellum, we show that the coordinated beating essentially responds to only load exerted on the cis flagellum; and that such asymmetry in response derives from a unilateral coupling between the two flagella. Our results highlight a distinct role for each flagellum in coordination and have implication for biflagellates tactic behaviors.

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield, while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real-time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating both larger droplets and, likely, GUV-sized satellite droplets. According to existing theoretical models, the oil-water interface should allow for crossing of all droplets, but based on our observations and theoretical modelling of the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE, but ultimately also aid the development of more reliable and efficient methods for GUV production.

A three-dimensional (3D) numerical simulation was performed using a combined stroke swimmer (deformable sphere) in an incompressible fluid of an infinite domain. The time-dependent deformation of the swimmer surface was assumed independent of the circumferential cross section in the flow direction of the swimmer. The 3D numerical simulation is an extension of our previous study that considered an axisymmetric numerical simulation. In particular, different fluid viscosities were considered for the same stroke of the swimmer. The effect of the swimmer inertia was studied by gradually decreasing the fluid viscosity. When the fluid viscosity decreased, the mean velocity of the swimmer changed its direction between Re = 0.00189 and Re = 0.0103. There is a transition between Re = 0.0103 and Re = 9.90 from the axisymmetric to three-dimensional flow that exhibits planar symmetry.

Vital biological processes, such as trafficking, sensing, and motility, are facilitated by cellular lipid membranes, which interact mechanically with surrounding fluids. Such lipid membranes are only a few nanometers thick and composed of a liquid crystalline structure known as the lipid bilayer. Here, we introduce an active, noncontact, two-point microrheology technique combining multiple optical tweezers probes with planar freestanding lipid bilayers accessible on both sides. We use the method to quantify both fluid slip close to the bilayer surface and transmission of fluid flow across the structure, and we use numerical simulations to determine the monolayer viscosity and the intermonolayer friction. We find that these physical properties are highly dependent on the molecular structure of the lipids in the bilayer. We compare ordered-phase with liquid disordered-phase lipid bilayers, and we find the ordered-phase bilayers to be 10 to 100 times more viscous but with 100 times less intermonolayer friction. When a local shear is applied by the optical tweezers, the ultralow intermonolayer friction results in full slip of the two leaflets relative to each other and as a consequence, no shear transmission across the membrane. Our study sheds light on the physical principles governing the transfer of shear forces by and through lipid membranes, which underpin cell behavior and homeostasis.

The accumulation of motile cells at solid interfaces increases the rate of surface encounters and the likelihood of surface attachment, leading to surface colonization and biofilm formation. The cell density distribution in the vicinity of a physical boundary is influenced by the interactions between the microswimmers and their physical environment, including hydrodynamic and steric interactions, as well as by stochastic effects. Disentangling the contributions of these effects remains an experimental challenge. Here, we use a custom-made four-camera view microscope to track a population of motile puller-type Chlamydomonas reinhardtii in a relatively unconstrained three-dimensional (3D) domain. Our experiments yield an extensive sample of 3D trajectories including cell-surface encounters with a planar wall, from which we extract a full description of the dynamics and the stochasticity of swimming cells. We use this large data sample and combine it with Monte Carlo simulations to determine the link between the dynamics at the single-cell level and the cell density. Our experiments demonstrate that the near-wall scattering is bimodal, corresponding to steric and hydrodynamic effects. We find, however, that this near-wall dynamics has little influence on the cell accumulation at the surface. On the other hand, we present evidence of a cell-induced surface-directed rotation leading to a vertical orbiting behavior and hopping trajectories, consistent with long-range hydrodynamic effects. We identify this long-range effect to be at the origin of the significant surface accumulation of cells.

The swift deformations of flagella and cilia are crucial for locomotion and fluid transport on the micron scale. Most hydrodynamic models of flagellar and ciliary flows assume the zero Reynolds number limit and model the flow using Stokes equations. Recent work has demonstrated that this quasi-steady approximation breaks down at increasing distances from the cilia. Here, we use optical tweezer-based velocimetry to measure the flow velocity with high temporal accuracy, and to reconstruct the entire unsteady flow field around beating cilia. We report both the steady and the unsteady component of the ciliary flow and compare them with the solutions to both the Stokes and the Navier-Stokes equations. Our experimental measurements of the velocity and vorticity fields are in agreement with the numerical solution to the Navier-Stokes equations and show significant differences with the solution to the Stokes equations. We characterize the phase difference between the flow oscillations and the oscillations of the ciliary motion and evidence a significant anisotropic phase lag. We show that this phase lag presents the spatiotemporal characteristics of the unsteady Stokes equations and that the flow field around beating cilia is well represented by the fundamental solution to the unsteady Stokes equations: the oscillet.

A numerical simulation has been made of the combined stroke swimmer (a deformable sphere) and compared with the results of the second-order perturbation theory of Felderhof and Jones (2017). At a small ratio of the amplitude of the deformation of the sphere and the radius of the sphere the numerical and theoretical results agree well. However for a larger value of this ratio the results deviate due to inertia. The streamlines, as calculated numerically, change significantly with increasing inertia.

The original version of this article contained a mistake.

In the study of micro-scale biological flows, velocimetry methods based on passive tracers, such as micro-PIV and micro-PTV, are well established to characterize steady flows. However, these methods become inappropriate for measuring unsteady flows of small amplitude, because, on these scales, the motion of passive tracers cannot be distinguished from Brownian motion. In this study, we use optical tweezers (OTs) in combination with Kalman filtering, to measure unsteady microscopic flows with high temporal accuracy. This method is referred to as optical tweezers-based velocimetry (OTV). The OTV method measures the nanometric displacements of a trapped bead, and predicts the instantaneous velocity of the flow by employing a Kalman filter. We discuss the accuracy of OTV in measuring unsteady flows with 1.5–70 $$\upmu$$ m s $$^{-1}$$ amplitudes and 10–90 Hz frequencies. We quantify how the bead size and the laser power affect the velocimetry accuracy, and specify the optimal choices for the bead size and laser power to measure different unsteady flows. OTV accurately measures unsteady flows with amplitudes as small as 3–6  $$\upmu$$ m s $$^{-1}$$ . We compare the accuracy of OTV and micro-PTV, and characterize the flow regime for which OTV outperforms micro-PTV. We also demonstrate the robustness of OTV by measuring the unsteady flow created by the cilia of green alga Chlamydomonas reinhardtii, and comparing with numerical predictions based on Stokes equations. An open-source implementation of the OTV software in Matlab is available through the 4TU.Centre for Research Data.

The flagella of Chlamydomonas reinhardtii possess fibrous ultrastructures of a nanometer-scale thickness known as mastigonemes. These structures have been widely hypothesized to enhance flagellar thrust; however, detailed hydrodynamic analysis supporting this claim is lacking. In this study, we present a comprehensive investigation into the hydrodynamic effects of mastigonemes using a genetically modified mutant lacking the fibrous structures. Through high-speed observations of freely swimming cells, we found the average and maximum swimming speeds to be unaffected by the presence of mastigonemes. In addition to swimming speeds, no significant difference was found for flagellar gait kinematics. After our observations of swimming kinematics, we present direct measurements of the hydrodynamic forces generated by flagella with and without mastigonemes. These measurements were conducted using optical tweezers, which enabled high temporal and spatial resolution of hydrodynamic forces. Through our measurements, we found no significant difference in propulsive flows due to the presence of mastigonemes. Direct comparison between measurements and fluid mechanical modeling revealed that swimming hydrodynamics were accurately captured without including mastigonemes on the modeled swimmer's flagella. Therefore, mastigonemes do not appear to increase the flagella's effective area while swimming, as previously thought. Our results refute the longstanding claim that mastigonemes enhance flagellar thrust in C. reinhardtii, and so, their function still remains enigmatic.