Embedded Microbubbles for Acoustic Manipulation of Single Cells and Microfluidic Applications.

Acoustically excited microstructures have demonstrated significant potential for small-scale biomedical applications by overcoming major microfluidic limitations. Recently, the application of oscillating microbubbles has demonstrated their superiority over acoustically excited solid structures due to their enhanced acoustic streaming at low input power. However, their limited temporal stability hinders their direct applicability for industrial or clinical purposes. Here, we introduce the embedded microbubble, a novel acoustofluidic design based on the combination of solid structures (poly(dimethylsiloxane)) and microbubbles (air-filled cavity) to combine the benefits of both approaches while minimizing their drawbacks. We investigate the influence of various design parameters and geometrical features through numerical simulations and experimentally evaluate their manipulation capabilities. Finally, we demonstrate the capabilities of our design for microfluidic applications by investigating its mixing performance as well as through the controlled rotational manipulation of individual HeLa cells.

Acoustic Radiation Forces Produced by Sharp-Edge Structures in Microfluidic Systems.

We study sharp-edge structures that are used in microfluidic systems for particle and cell manipulation. Experiments show that oscillating sharp edges can attract or repel particles suspended in a microfluidic channel. This effect is caused by acoustic radiation forces induced by sharp edges. We propose an analytical theory that allows one to evaluate the acoustic radiation force produced by a sharp-edge structure on elastic particles and to study which parameters govern the interaction of particles with a sharp-edge structure, forcing them to be attracted in one situation and to be repelled in another situation. The proposed theory gives foundations for the design of microfluidic systems making use of sharp edges for particle trapping. We also provide experimental data to validate the theory.

Acoustic Compressibility of Caenorhabditis elegans.

The acoustic compressibility of Caenorhabditis elegans is a necessary parameter for further understanding the underlying physics of acoustic manipulation techniques of this widely used model organism in biological sciences. In this work, numerical simulations were combined with experimental trajectory velocimetry of L1 C. elegans larvae to estimate the acoustic compressibility of C. elegans. A method based on bulk acoustic wave acoustophoresis was used for trajectory velocimetry experiments in a microfluidic channel. The model-based data analysis took into account the different sizes and shapes of L1 C. elegans larvae (255 ± 26 µm in length and 15 ± 2 µm in diameter). Moreover, the top and bottom walls of the microfluidic channel were considered in the hydrodynamic drag coefficient calculations, for both the C. elegans and the calibration particles. The hydrodynamic interaction between the specimen and the channel walls was further minimized by acoustically levitating the C. elegans and the particles to the middle of the measurement channel. Our data suggest an acoustic compressibility κCe of 430 TPa-1 with an uncertainty range of ±20 TPa-1 for C. elegans, a much lower value than what was previously reported for adult C. elegans using static methods. Our estimated compressibility is consistent with the relative volume fraction of lipids and proteins that would mainly make up for the body of C. elegans. This work is a departing point for practical engineering and design criteria for integrated acoustofluidic devices for biological applications.

Acoustofluidic particle dynamics: Beyond the Rayleigh limit.

In this work a numerical model to calculate the trajectories of multiple acoustically and hydrodynamically interacting spherical particles is presented. The acoustic forces are calculated by solving the fully coupled three-dimensional scattering problem using finite element software. The method is not restricted to single re-scattering events, mono- and dipole radiation, and long wavelengths with respect to the particle diameter, thus expanding current models. High frequency surface acoustic waves have been used in the one cell per well technology to focus individual cells in a two-dimensional wave-field. Sometimes the cells started forming clumps and it was not possible to focus on individual cells. Due to a lack of existing theory, this could not be fully investigated. Here, the authors use the full dynamic simulations to identify limiting factors of the one-cell-per-well technology. At first, the authors demonstrate good agreement of the numerical model with analytical results in the Rayleigh limiting case. A frequency dependent stability exchange between the pressure and velocity was then demonstrated. The numerical formulation presented in this work is relatively general and can be used for a multitude of different high frequency applications. It is a powerful tool in the analysis of microscale acoustofluidic devices and processes.

Multibody dynamics in acoustophoresis.

Determining the trajectories of multiple acoustically and hydrodynamically interacting as well as colliding particles is one of the challenges in numerical acoustophoresis. Although the acoustic forces between multiple small spherical particles can be obtained analytically, previous research did not address the particle-particle contacts in a rigorous way. This article extends existing methods by presenting an algorithm on displacement level which models the hard contacts using set-valued force laws, hence allowing for the first time the computation of a first approximation of complete trajectories of multiple hydrodynamically and acoustically interacting particles. This work uses a semi-analytical method to determine the acoustic forces, which is accurate up to the dipole contributions of the multipole expansion. The hydrodynamic interactions are modeled using the resistance and mobility functions of the Stokes' flow. In previous experimental work particles have been reported to interact acoustically, ultimately forming stacked lines near the pressure nodes of a standing wave. This phenomenon is examined experimentally and numerically, the simulation shows good agreement with the experimental results. To demonstrate the capabilities of the method, the rotation of a particle clump in two orthogonal waves is simulated. The presented method allows further insight in self-assembly applications and acoustic particle manipulation.

Artificial Swimmers Propelled by Acoustically Activated Flagella.

Recent studies have garnered considerable interest in the field of propulsion to maneuver micro- and nanosized objects. Acoustics provide an alternate and attractive method to generate propulsion. To date, most acoustic-based swimmers do not use structural resonances, and their motion is determined by a combination of bulk acoustic streaming and a standing-wave field. The resultant field is intrinsically dependent on the boundaries of their resonating chambers. Though acoustic based propulsion is appealing in biological contexts, existing swimmers are less efficient, especially when operating in vivo, since no predictable standing-wave can be established in a human body. Here we describe a new class of nanoswimmer propelled by the small-amplitude oscillation of a flagellum-like flexible tail in standing and, more importantly, in traveling acoustic waves. The artificial nanoswimmer, fabricated by multistep electrodeposition techniques, compromises a rigid bimetallic head and a flexible tail. During acoustic excitation of the nanoswimmer the tail structure oscillates, which leads to a large amplitude propulsion in traveling waves. FEM simulation results show that the structural resonances lead to high propulsive forces.

Viscous torque on spherical micro particles in two orthogonal acoustic standing wave fields.

This paper reports the experimental results of the acoustic rotation of spherical micro particles because of two orthogonal standing waves. When the standing waves are excited at equal frequency but with a phase shift between two external voltage signals there is an acoustic streaming around the particles. This streaming is due to a time averaging of the acoustic wave field and produces a nonzero viscous torque on the particles, driving them to rotate. The work investigates the micro-particle rotation due to the viscous torque and predict the particle's steady state rotational velocity. The previous theoretical discussions [Nyborg, J. Acoust. Soc. Am. 85, 329-339 (1958); Lee and Wang, J. Acoust. Soc. Am. 85, 1081-1088 (1989)] of the viscous torque on a non-rotating sphere are expanded to allow free rotations. The analytical calculations provide a deeper understanding of the viscous torque and explain the experimental observations of rotating particles. A macroscopic experimental device is designed to provide the necessary boundary conditions for the viscous torque to rotate spherical particles. The experiments not only show good agreement with the analysis, but also demonstrate that the viscous torque due to acoustic streaming may dominate for the case of near-spherical particle dynamics.

Acoustophoresis of disk-shaped microparticles: A numerical and experimental study of acoustic radiation forces and torques.

Disk-shaped microparticles experience an acoustic radiation force and torque in an ultrasonic standing wave. Hence, they are translated by the acoustic field, an effect called acoustophoresis, and rotated. The torque effect is also known from the "Rayleigh disk" which is described in literature for sound intensity measurements. In this paper, inviscid numerical simulations of acoustic radiation forces and torques for disks with radius ≪ wavelength in water are developed in good agreement with former analytical solutions, and the dependence on disk geometry, density, and orientation is discussed. Experiments with alumina disks (diameter 7.5 µm), suspended in an aqueous liquid in a silicon microchannel, confirm the theoretical results qualitatively at the microscale and ultrasonic frequencies around 2 MHz. These results can potentially be applied for the synthesis of disk-reinforced composite materials. The insights are also relevant for the acoustic handling of various disk-shaped particles, such as red blood cells.

Numerical investigation of crack propagation regimes in snow fracture experiments.

A snow slab avalanche releases after failure initiation and crack propagation in a highly porous weak snow layer buried below a cohesive slab. While our knowledge of crack propagation during avalanche formation has greatly improved over the last decades, it still remains unclear how snow mechanical properties affect the dynamics of crack propagation. This is partly due to a lack of non-invasive measurement methods to investigate the micro-mechanical aspects of the process. Using a DEM model, we therefore analyzed the influence of snow cover properties on the dynamics of crack propagation in weak snowpack layers. By focusing on the steady-state crack speed, our results showed two distinct fracture process regimes that depend on slope angle, leading to very different crack propagation speeds. For long experiments on level terrain, weak layer fracture is mainly driven by compressive stresses. Steady-state crack speed mainly depends on slab and weak layer elastic moduli as well as weak layer strength. We suggest a semi-empirical model to predict crack speed, which can be up to 0.6 times the slab shear wave speed. For long experiments on steep slopes, a supershear regime appeared, where the crack propagation speed reached approximately 1.6 times the slab shear wave speed. A detailed micro-mechanical analysis of stresses revealed a fracture principally driven by shear. Overall, our findings provide new insight into the micro-mechanics of dynamic crack propagation in snow, and how these are linked to snow cover properties. Supplementary Information: The online version contains supplementary material available at 10.1007/s10035-024-01423-5.

Schlieren visualization of ultrasonic standing waves in mm-sized chambers for ultrasonic particle manipulation.

BACKGROUND: For the design and characterization of ultrasonic particle manipulation devices the pressure field in the fluid cavity is of great interest. The schlieren method provides an optical tool for the visualization of such pressure fields. Due to its purely optical nature this experimental method has got some unique advantages compared to methods like particle tracking. RESULTS: A vertical schlieren setup and an investigation with the same of a mm-sized chamber used to agglomerate particles are presented here. The schlieren images show a two-dimensional representation of the whole pressure distribution recorded simultaneously with a good resolution in time. The gained description of the pressure field is shown to be in agreement with a numerical simulation. Thermal effects as well as streaming effects are shown. CONCLUSIONS: The results show the great potential of schlieren visualization to investigate ultrasonic particle manipulation devices. Visualized are pressure fields, acoustic streaming, temperature effects and effects caused by fluid volumes of different density.

Rotation of non-spherical micro-particles by amplitude modulation of superimposed orthogonal ultrasonic modes.

Contactless rotation of non-spherical particles has been modeled and experimentally achieved using ultrasonic manipulation. For this purpose an acoustic radiation torque was generated by a time-varying pressure field resulting in a change of orientation of the potential well. The rotation method is based on amplitude modulation of two orthogonal ultrasonic modes. The force potential field has been used to evaluate the different modes and actuations to achieve rotation. Experiments have been performed in micro devices with copolymer particles and glass fibers at frequencies in the megahertz range. A continuous rotation was successfully demonstrated and the method allowed to stop the rotation at arbitrary angular positions.

Measuring the effects of a pulsed excitation on the buildup of acoustic streaming and the acoustic radiation force utilizing an optical tweezer.

Pulsed excitations of piezoelectric transducers affect during the buildup the force contributions from acoustic streaming (AS) and the acoustic radiation force (ARF) to the total force in a standing pressure wave differently. We find with an optical tweezer as measuring instrument that during the first 120 000 excitation periods and across different pulsing frequencies, the AS-induced displacement is on average less than 20% of its nonpulsed value for a duty cycle of 50%, whereas the ARF-induced displacement is around 50%. These findings show that a pulsed excitation can be a tool for reducing AS compared to the ARF.

Influence of particle shape and material on the acoustic radiation force and microstreaming in a standing wave.

In view of its influence on the acoustic radiation force, we investigate the microstreaming around a small solid elastic particle in an ultrasonic standing wave in dependence of its material properties and shape. The configuration is axisymmetric, making it accessible to numerical methods, such as the finite element method. The results reveal a transition from viscous scattering- to microstreaming-dominated acoustic radiation force that depends on the particle density. When a deviation of the particle shape from a sphere becomes smaller than the viscous boundary layer thickness, we show that the influence of the shape on the viscous contributions to the acoustic radiation force diminishes, allowing the use of theoretical models for a spherical particle. However, extreme asymmetric shape perturbations, such as crowns with sharp edges, can give rise to noticeable viscous contributions for a dense particle that is larger than the viscous boundary layer thickness. We also introduce a hybrid analytical model for the acoustic radiation force on a spherical particle that accounts for the microstreaming and particle compressibility and shows a good agreement with numerical simulations for an arbitrary particle size and density.

Longevity interventions temporally scale healthspan in Caenorhabditis elegans.

Human centenarians and longevity mutants of model organisms show lower incidence rates of late-life morbidities than the average population. However, whether longevity is caused by a compression of the portion of life spent in a state of morbidity, i.e., "sickspan," is highly debated even in isogenic Caenorhabditis elegans. Here, we developed a microfluidic device that employs acoustophoretic force fields to quantify the maximum muscle strength and dynamic power in aging C. elegans. Together with different biomarkers for healthspan, we found a stochastic onset of morbidity, starting with a decline in dynamic muscle power and structural integrity, culminating in frailty. Surprisingly, we did not observe a compression of sickspan in longevity mutants but instead observed a temporal scaling of healthspan. Given the conservation of these longevity interventions, this raises the question of whether the healthspan of mammalian longevity interventions is also temporally scaled.

Interparticle attraction along the direction of the pressure gradient in an acoustic standing wave.

Scattering of an acoustic wave by particles gives rise to microstreaming, as well as to acoustic radiation and interaction forces on the particles. We numerically study these steady, nonlinear phenomena for a case of two elastic spheres in a standing wave. We show that if one or both spheres are smaller or comparable to the viscous boundary layer, the microstreaming close to the pressure node can lead to an interparticle attraction along the direction of the pressure gradient of the wave. Similar behavior is observed when, instead of size, density of one of the spheres is sufficiently larger relative to the other sphere. These findings could promote the acoustic manipulation of nanoparticles and bacteria.

Tools for manipulation and positioning of microtissues.

Complex three-dimensional (3D) in vitro models are emerging as a key technology to support research areas in personalised medicine, such as drug development and regenerative medicine. Tools for manipulation and positioning of microtissues play a crucial role in the microtissue life cycle from production to end-point analysis. The ability to precisely locate microtissues can improve the efficiency and reliability of processes and investigations by reducing experimental time and by providing more controlled parameters. To achieve this goal, standardisation of the techniques is of primary importance. Compared to microtissue production, the field of microtissue manipulation and positioning is still in its infancy but is gaining increasing attention in the last few years. Techniques to position microtissues have been classified into four main categories: hydrodynamic techniques, bioprinting, substrate modification, and non-contact active forces. In this paper, we provide a comprehensive review of the different tools for the manipulation and positioning of microtissues that have been reported to date. The working mechanism of each technique is described, and its merits and limitations are discussed. We conclude by evaluating the potential of the different approaches to support progress in personalised medicine.

Optical feedback control loop for the precise and robust acoustic focusing of cells, micro- and nanoparticles.

Despite a long history and the vast number of applications demonstrated, very few market products incorporate acoustophoresis. Because a human operator must run and control a device during an experiment, most devices are limited to proof of concepts. On top of a possible detuning due to temperature changes, the human operator introduces a bias which reduces the reproducibility, performance and reliability of devices. To mitigate some of these problems, we propose an optical feedback control loop that optimizes the excitation frequency. We investigate the improvements that can be expected when a human operator is replaced for acoustic micro- and nanometer particle focusing experiments. Three experiments previously conducted in our group were taken as a benchmark. In addition to being automatic, this resulted in the feedback control loop displaying a superior performance compared to an experienced scientist in 1) improving the particle focusing by at least a factor of two for 5 µm diameter PS particles, 2) increasing the range of flow rates in which 1 µm diameter PS particles could be focused and 3) was even capable of focusing 600 nm diameter PS particles at a frequency of 1.72075 MHz. Furthermore, the feedback control loop is capable of focusing biological cells in one and two pressure nodes. The requirements for the feedback control loop are: an optical setup, a run-of-the-mill computer and a computer controllable function generator. Thus resulting in a cost-effective, high-throughput and automated method to rapidly increase the efficiency of established systems. The code for the feedback control loop is openly accessible and the authors explicitly wish that the community uses and modifies the feedback control loop to their own needs.

Theoretical and numerical calculations for the time-averaged acoustic force and torque acting on a rigid cylinder of arbitrary size in a low viscosity fluid.

In this paper, theoretical calculations as well as numerical simulations are performed for the time-averaged acoustic force and torque on a rigid cylinder of arbitrary size in a fluid with low viscosity, i.e., the acoustic boundary layer is thin compared to the cylinder radius. An exact analytical solution and its approximation are proposed in the form of an infinite series including Bessel functions. These solutions can be evaluated easily by a mathematical software package such as mathematica and matlab. Three types of incident waves, plane traveling wave, plane standing wave, and dual orthogonal standing waves, are investigated in detail. It is found that for a small particle, the viscous effects for an incident standing wave may be neglected but those for an incident traveling wave are notable. A nonzero viscous torque is experienced by the rigid cylinder when subjected to dual orthogonal standing waves with a phase shift even when the cylinder is located at equilibrium positions without imposed acoustic forces. Furthermore, numerical simulations are carried out based on the FVM algorithm to verify the proposed theoretical formulas. The theoretical results and the numerical ones agree with each other very well in all the cases considered.

Cleaning ability and induced dentin loss of a magnetostrictive ultrasonic instrument at different power settings.

Some laboratory studies have evaluated the oscillation mode of ultrasonic scalers. None of them recorded its influence on calculus removal and quantified dental hard tissue loss. This study aimed to compare the performance of a magnetostrictive ultrasonic instrument at different power settings in vitro in relation to the tip oscillation activity. The oscillation activity of the straight Slimline® insert in the Cavitron® ultrasonic scaling device was analyzed at five different power settings with the help of two laser vibrometers. The performance of this instrument was tested on 60 roots of human single-rooted teeth. Twelve roots each were randomly assigned to be instrumented at a given power setting. Every root was instrumented for 120 s at a standardized instrumentation force of 0.1 ± 0.05 N. In addition, another 30 periodontally involved roots with subgingival calculus were instrumented accordingly to assess the calculus removal potential. The surface characteristics after instrumentation were analyzed under scanning electron microscope. The instrumentation at minimum power setting resulted in an mean increase of the root surface roughness of 0.18 ± 0.28 compared to 0.51 ± 0.48 at maximum power setting (P = 0.0327). The loss of dental hard tissue amounted to 11.37 ± 3.64 at minimum compared to 23.37 ± 15.76 at maximum power (P = 0.0010). The higher the power setting, the more calculus was removed. The values of the latter ranged between 4.04 ± 1.87 and 11.26 ± 4.66 mm² of cleaned dentin surface area (P = 0.0065). At lower power settings, a more favorable relation between cleaning ability, loss of dentine, and surface roughness was found.

Dynamic measurement of the acoustic streaming time constant utilizing an optical tweezer.

The combination of a bulk acoustic wave device and an optical trap allows for studying the buildup time of the respective acoustic forces. In particular, we are interested in the time it takes to build up the acoustic radiation force and acoustic streaming. For that, we measure the trajectory of a spherical particle in an acoustic field over time. The shape of the trajectory is determined by the acoustic radiation force and by acoustic streaming, both acting on different time scales. For that, we utilize the high temporal resolution (Δt=0.8µs) of an optical trapping setup. With our experimental parameters the acoustic radiation force on the particle and the acoustic streaming field theoretically have characteristic buildup times of 1.4µs and 1.44ms, respectively. By choosing a resonance mode and a measurement position where the acoustic radiation force and acoustic streaming induced viscous drag force act in orthogonal directions, we can measure the evolution of these effects separately. Our results show that the particle is accelerated nearly instantaneously by the acoustic radiation force to a constant velocity, whereas the acceleration phase to a constant velocity by the acoustic streaming field takes significantly longer. We find that the acceleration to a constant velocity induced by streaming takes in average about 17 500 excitation periods (≈4.4ms) longer to develop than the one induced by the acoustic radiation force. This duration is about four times larger than the so-called momentum diffusion time which is used to estimate the streaming buildup. In addition, this rather large difference in time can explain why a pulsed acoustic excitation can indeed prevent acoustic streaming as it has been shown in some previous experiments.