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.

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 × 2.5 × 2 mm3. 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.

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 μm-sized droplets and only in some cases GUV-sized (∼15 μm) 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.

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.

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.

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.

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.

Manipulating particles is of interest in diverse fields of engineering. Generally, manipulation activities carried out in micro-devices have a fixed design tailored to specific task. To address this issue, we designed a Hele-Shaw flow cell with "virtual" channels generated by uniform flow in transverse direction and three inlets in the longitudinal axis. These three inlets can inject or dispense fluid in the flow cell to deviate the streamlines. This device provides us the opportunity to integrate multiple functionalities such as particle trapping and separation onto a single device. Since the depth-averaged velocity over the channel in a Hele-Shaw cell is irrotational, we use potential flow theory to predict the flow field for manipulating particles.

Abstract: 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 μ 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 μ 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. Graphic abstract: [Figure not available: see fulltext.].

Abstract: Obtaining accurate experimental data from Lagrangian tracking and tomographic velocimetry requires an accurate camera calibration consistent over multiple views. Established calibration procedures are often challenging to implement when the length scale of the measurement volume exceeds that of a typical laboratory experiment. Here, we combine tools developed in computer vision and non-linear camera mappings used in experimental fluid mechanics, to successfully calibrate a four-camera setup that is imaging inside a large tank of dimensions ∼10×25×6m3. The calibration procedure uses a planar checkerboard that is arbitrarily positioned at unknown locations and orientations. The method can be applied to any number of cameras. The parameters of the calibration yields direct estimates of the positions and orientations of the four cameras as well as the focal lengths of the lenses. These parameters are used to assess the quality of the calibration. The calibration allows us to perform accurate and consistent linear ray-tracing, which we use to triangulate and track fish inside the large tank. An open-source implementation of the calibration in Matlab is available. Graphic abstract: [Figure not available: see fulltext.].

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.

Abstract: In microscopic particle image velocimetry (micro-PIV), correlation averaging over multiple frames is often required, leading to a loss in temporal resolution, therefore limiting the measurement accuracy for unsteady flows. Here, we present a new PIV method suitable to study steady and unsteady laminar flows between parallel plates (i.e., Hele-Shaw flow), which is a common flow configuration in microfluidic applications. Our method reduces the effective seeding density and yields similar if not higher signal-to-noise ratio (SNR) compared to conventional micro-PIV. We call this algorithm Ψ -PIV. Ψ -PIV requires a much smaller number of frames to reach the same SNR compared to the widely used correlation averaging method. This leads to a significant improvement of the temporal resolution. The Ψ -PIV algorithm is used in an experimental investigation of steady and unsteady flows in a Hele-Shaw cell. Our experiment shows that Ψ -PIV reduces the number of required frames by 8 times and 30 times compared to the frames required by conventional PIV for steady and unsteady laminar flow, respectively. In this study, PIV and Ψ -PIV use a single-pass cross-correlation to present the underlying difference between the two approaches. Graphic abstract: [Figure not available: see fulltext.].

We detail the analysis of centrifugal homogenization process by a hydrodynamic model and the model-guided design of a low-cost centrifugal homogenizer. During operation, centrifugal force pushes a multiphase solution to be homogenized through a thin nozzle, consequently homogenizing its contents. We demonstrate and assess the homogenization of coarse emulsions into relatively monodisperse emulsions, as well as the application of centrifugal homogenization in the mechanical lysis of mpkCCD mouse kidney cells. To gain insight into the homogenization mechanism, we investigate the dependence of emulsion droplet size on geometrical parameters, centrifugal acceleration, and dispersed phase viscosity. Our experimental results are in qualitative agreement with models predicting the droplet size. Furthermore, they indicate that high shear rates kept constant throughout operation produce more monodisperse droplets. We show this ideal homogenization condition can be realized through hydrodynamic model-guided design minimizing transient effects inherent to centrifugal homogenization. Moreover, we achieved power densities comparable to commercial homogenizers by model guided optimization of homogenizer design and experimental conditions. Centrifugal homogenization using the proposed homogenizer design thus offers a low-cost alternative to existing technologies as it is constructed from off-the-shelf parts (Falcon tubes, syringe, needles) and used with a centrifuge, readily available in standard laboratory environment.

Cell lipid membranes are the site of vital biological processes, such as motility, trafficking, and sensing, many of which involve mechanical forces. Elucidating the interplay between such bioprocesses and mechanical forces requires the use of tools that apply and measure piconewton-level forces, e.g., optical tweezers. Here, we introduce the combination of optical tweezers with free-standing lipid bilayers, which are fully accessible on both sides of the membrane. In the vicinity of the lipid bilayer, optical trapping would normally be impossible due to optical distortions caused by pockets of the solvent trapped within the membrane. We solve this by drastically reducing the size of these pockets via tuning of the solvent and flow cell material. In the resulting flow cells, lipid nanotubes are straightforwardly pushed or pulled and reach lengths above half a millimeter. Moreover, the controlled pushing of a lipid nanotube with an optically trapped bead provides an accurate and direct measurement of important mechanical properties. In particular, we measure the membrane tension of a free-standing membrane composed of a mixture of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) to be 4.6 × 10-6 N/m. We demonstrate the potential of the platform for biophysical studies by inserting the cell-penetrating trans-activator of transcription (TAT) peptide in the lipid membrane. The interactions between the TAT peptide and the membrane are found to decrease the value of the membrane tension to 2.1 × 10-6 N/m. This method is also fully compatible with electrophysiological measurements and presents new possibilities for the study of membrane mechanics and the creation of artificial lipid tube networks of great importance in intra- and intercellular communication.

Stokes equations are commonly used to model the hydrodynamic flow around cilia on the micron scale. The validity of the zero Reynolds number approximation is investigated experimentally with a flow velocimetry approach based on optical tweezers, which allows the measurement of periodic flows with high spatial and temporal resolution. We find that beating cilia generate a flow, which fundamentally differs from the stokeslet field predicted by Stokes equations. In particular, the flow velocity spatially decays at a faster rate and is gradually phase delayed at increasing distances from the cilia. This indicates that the quasisteady approximation and use of Stokes equations for unsteady ciliary flow are not always justified and the finite timescale for vorticity diffusion cannot be neglected. Our results have significant implications in studies of synchronization and collective dynamics of microswimmers.