Acoustical tweezers can manipulate inanimate particles as well as living cells in liquid in 2D using surface acoustic waves and in 3D using bulk acoustic waves. Here, we demonstrate a MHz twin-trap device for underwater manipulation of particles and cell aggregates and show that it operates effectively within a Petri dish and a plastic tube. We also describe a method to visualize in real time the acoustic field using a contained layer of small, high-density particles, which allows the trap to be seen in the same image as the manipulated particle. The device used was made with simple components, integrated onto a printed circuit board, and requires a single electrical channel for excitation. As a result, this device has the potential to be widely implemented in applications such as micro-organism manipulation, in vivo manipulation, and drug delivery.

Acoustical tweezing has become of interest in the scientific community and has served as a tool in several branches of science, such as biology,1–5 medicine,6–10 manufacturing,11–14 and chemistry.13,15–18

Acoustic manipulation technology has also been conceived as a feasible method for container-less processing in manufacturing.19 Diverse ways to achieve acoustic particle manipulation have been reported, including 2D devices based on surface acoustic waves (SAWs)20–24 and 3D beam devices25–30 in which trapping fields are formed using holograms29,31,32 or arrays of transducers.27,33–39

Acoustical tweezers share much in common with optical tweezers that use the momentum carried by light waves.40,41 However, for the same power input, the forces generated in acoustical tweezers are five orders of magnitude greater.42 In addition, acoustical tweezers have generally shown high biocompatibility.10,43,44 Magnetic forces have also been used to manipulate biological samples,45 but these have the disadvantage of requiring the addition of functionalized magnetic tags.46 

A type of acoustical tweezer known as a single beam twin-trap has been reported to manipulate particles through air47 and liquid,48 where the latter is particularly important for the biomedical field.49 In this paper, a high frequency (MHz) single beam twin-trap device is described, in which the trap is generated by a transducer pair operating out of phase, combined with a focusing Fresnel lens.

More specifically, this paper describes a 1.76 MHz device that demonstrates 3D manipulation of particles (levitation), and also manipulation of cell aggregates through structures; additionally, this work also describes a 4.32 MHz device that demonstrates manipulation on the cellular length scale. Like the more widely explored vortex traps,26,38,50–53 the single beam twin-trap also results in high selectivity, as the trap can be focused into a specific local region.54 A twin-trap results in potential energy minimum at the focus and a 3D trap. In contrast to a vortex-trap, which has a ring-like high-intensity pressure region surrounding a low-intensity region at the center of the trap at its focal plane, a twin-trap has two lobe-like high-intensity pressure regions and a low-pressure region in between. Because of this, a twin-trap maximizes the trapping force in the direction perpendicular to the two high-intensity pressure lobes. Importantly, twin-trapping allows device designs to be simplified29 leading to inexpensive components and simple experimental setup, compared to vortex trapping, which require complex printed micro-electro-mechanical systems26 or multiple-element arrays.55 

Acoustic manipulation has become difficult due to the lack of methods to simultaneously visualize both the object under manipulation and the acoustic field, raising challenges in application development. In this paper, a method to visualize the acoustic field in real time is described. This aids the exploration of interesting applications as shown here such as moving clusters of cells within a Petri dish and moving particles within a small plastic tube.

The twin-trap setup shown in Fig. 1(a) uses the acoustic radiation force56 that acts on particles within an acoustic field. The device consists of two piezoelectric transducers that are excited out-of-phase such that a plane of cancelation is created between them. This transducer pair was manufactured by dicing a circular piezoceramic disk into two parts and making separate connections to the resulting elements. The transducer pair was attached to a 3D printed Fresnel lens that causes a focusing effect. The PCB was designed in such way that the out-of-phase connection between the piezoelectric elements was possible as seen in Fig. 1(b). This combination of a plane of cancelation, causing a zero-pressure plane, and focusing, results in the formation of a twin-trap at the focus. We introduce a visualization layer, which made the twin-trap lobes visible at the focus, as seen in Fig. 1(c).

FIG. 1.

Experimental details. (a) The experiment setup that consists of an electrical connection to a PCB board onto which the transducers and Fresnel lens are mounted. The Petri dish containing the particles under manipulation is mounted on a 3-axis manipulation stage. The visualization layer is positioned directly below the particles. The manipulation process is observed with a camera and illumination from above. (b) The electrical connection to the transducers. (c) A 130 μm diameter polyethylene particle is trapped at the center of a 1.76 MHz twin-trap, with the two high-pressure lobes of the trap observed either side using the visualization layer. Scale bar is 1 mm.

FIG. 1.

Experimental details. (a) The experiment setup that consists of an electrical connection to a PCB board onto which the transducers and Fresnel lens are mounted. The Petri dish containing the particles under manipulation is mounted on a 3-axis manipulation stage. The visualization layer is positioned directly below the particles. The manipulation process is observed with a camera and illumination from above. (b) The electrical connection to the transducers. (c) A 130 μm diameter polyethylene particle is trapped at the center of a 1.76 MHz twin-trap, with the two high-pressure lobes of the trap observed either side using the visualization layer. Scale bar is 1 mm.

Close modal

Figure 2(a) shows a Huygens' principle simulation of the acoustic pressure field in the focal plane (i.e., x-y plane for z = 20 mm) of a 1.76 MHz (transducer diameter 20 mm and focal length 20 mm) twin-trap from which its two characteristic high pressure-amplitude lobes can be seen. Figure 2(c) shows a further simulation revealing the acoustic pressure distribution in the x-z plane (for y = 0), and here, the effect of the focusing is evident. The overall effect here is to create a convergent 3D force field at the focus.29 

FIG. 2.

Acoustic pressure fields of the 1.76 MHz twin-trap device. Acoustic pressure in the x-y plane for z = 20 mm at the focus with (a) a Huygens' principle model simulation and (b) experimental measurement. Simulated acoustic pressure in the x-z plane for y = 0 is shown in (c) and the experimental equivalent in (d). The dotted circles in (a) and (c) indicate the position of the mica particle agglomeration within the visualization layer, which indicate the position of the twin trap. The distance between the two high amplitude zones (also called spatial selectivity54) of the twin-trap was measured as 1.7 mm, which is approximately two wavelengths in size.

FIG. 2.

Acoustic pressure fields of the 1.76 MHz twin-trap device. Acoustic pressure in the x-y plane for z = 20 mm at the focus with (a) a Huygens' principle model simulation and (b) experimental measurement. Simulated acoustic pressure in the x-z plane for y = 0 is shown in (c) and the experimental equivalent in (d). The dotted circles in (a) and (c) indicate the position of the mica particle agglomeration within the visualization layer, which indicate the position of the twin trap. The distance between the two high amplitude zones (also called spatial selectivity54) of the twin-trap was measured as 1.7 mm, which is approximately two wavelengths in size.

Close modal

The experimental setup is shown in Fig. 1(a) (see supplementary material, Fig. S1 for further details). A signal generator (33250A, Agilent, USA) and an amplifier (75A250A, Amplifier Research, USA) were connected to the piezoelectric elements via a USB connector linked to a circular magnetic and waterproof connector (8.5 mm diameter, Pogo-type). The sinusoidal electrical input was monitored on an oscilloscope (Keysight 33110A, Agilent, USA) and was typically 15–45  V pp.

The piezoelectric elements (PZT-5, Beijing Ultrasonic, China) were attached to a printed circuit board (PCB) using connection pads and bonded with conductive epoxy adhesive (8331D, MG Chemicals). A single drive channel can be used, and the phase reversal achieved by wiring the transducers with opposing polarity. One transducer disk had a thickness of 1.1 mm and a diameter 20 mm, while a thinner version had a thickness of 0.45 mm and a diameter of 10 mm. The piezoelectric transducers were diced into two equal semicircular pieces and covered with a silver wraparound electrode. The resonant frequency of the transducer elements was determined by electrical impedance measurements. The 1.1 mm thick transducer was found to have its first through-thickness resonance frequency at 1.76 MHz when attached to the PCB and the 0.45 mm thick transducer was found to be resonant at 4.32 MHz. Flexible adhesive was added to the cables attaching them to the cantilever device holder to prevent unwanted cable motion. A bubble level indicator was attached into the PCB to gauge flatness.

The Fresnel lenses were designed to delay the sound from some regions relative to others such that a focus was achieved at the desired focal length, z = 20 mm. Like any lens, the material used resulted in a pattern of phase delays, and the unique feature of the Fresnel lens is that this delay is modulo 2π, hence the well-known saw-tooth-like surface. The frequency, focal length, and diameter set the geometry of the lens, which was then printed using a resin 3D printer (Formlabs 2, Formlabs, USA) using a transparent resin (Clear FLGPCL02, Formlabs, USA). Inevitably, the 3D printed Fresnel lens had minor geometrical differences from the designed profile, and the profile of the printed lens was measured by slicing a lens and measuring the dimensions from a calibrated image. Finite element modeling (COMSOL Multiphysics, Version 6.1) was used to compare the acoustic field resulting from the printed lens relative to that from the original design, and this revealed a 1% difference in maximum focal pressure amplitude (see supplementary material, Fig. S2, for further details). The printed lenses were bonded to the piezoelectric transducers with an epoxy adhesive.

Particles were added to a polystyrene Petri dish (base thickness of 0.6 mm and a diameter of 3 cm) (Delsen, USA). A cantilever arm was used to position the Petri dish in the focal plane and move it in three axes, using a millimetric positioning stage (X-LSM050A-E03-PTB2, Zaber, Canada). A position-adjustable microscope camera (Sony IMX377 sensor, Japan) was located above the particles with a 130× magnification lens, as seen in Fig. 1(a).

The visualization layer that was attached to the Petri dish consisted of a container filled with a mixture of mica powder and water. This solution was made by adding 100 mL of water to 0.05 g of mica powder (available in a range of colors for use in craft projects) as well as three drops of liquid soap (ECOVER, UK) to prevent particles sticking to the walls of the container. The container itself was manufactured from a 3D printed ring of PLA (Ultimaker 2+, Ultimaker, Netherlands) of the same diameter as the Petri dish, bonded with flexible adhesive to two transparent 240 μm thick acetate layers. The distance between the two acetate layers was 2 mm. A small metal bar was positioned inside the visualization layer container to enable stirring of the particles from outside of the container with a magnet. This stirring was necessary since the mica particles tended to sediment with time.

The experiments were conducted by driving the signal generator at the resonant frequency of the piezoelectric transducers, i.e., 1.76 MHz, and using 30  V pp. For the higher frequency device working at 4.32 MHz, a voltage of 15  V pp was used. Both the amplifier and the signal generator signals were monitored by an oscilloscope. The twin-trap device was moved by either a motorized or manual 3-axis positioning stage.

The particle was initially positioned using a syringe. When the amplifier and the signal generator were turned on, the twin-trap lobes became visible through agglomeration of the mica particles in the visualization layer, allowing an easy positioning of the trap using the positioning stage, as shown in Fig. 1(c) (see the supplementary material for further detail).

All the particles considered here are simultaneously of high mass density and compressibility with respect to the surrounding water. Hence, according to Gor'kov's potential theory,57 if they are also small relative to the wavelength, then they can be expected to migrate away from the antinodes and toward the nodes of pressure. This nodal trapping can be seen considering the trapped particle in Fig. 1(c) and the pressure field around it in Figs. 2(a) and 2(c). Here, the polyethylene particle (density of 1 ± 0.01  g / mm 3, c = 2460 m/s, Cospheric, California, USA) is 130 μm in diameter relative to a wavelength in water of 851 μm. The visualization layer also uses relatively dense and stiff particles, and hence, the particles can be expected to move to the nodes. However, the layer allows visualization of the antinodes of the twin-trap as its surface acts as a reflector creating a standing wave with an amplitude distribution that matches the pressure field in the x-y plane (see the supplementary material videos for reference). The layer was set to be larger than a wavelength meaning that nodes were present just below the antinodes that formed the lobes of the twin-trap.

Figure 3 shows a demonstration of the manipulation of a single polyethylene 130 μm particle in three dimensions (Multimedia view). The particle was placed in the twin-trap using a syringe and manipulated over a distance of 6 mm in free-space in both the x and y axes. To manipulate the particle in the z-axis, the transducer was tilted manually by 45° with respect to the z-y plane to avoid reflections from the surface that causes some standing wave behavior in the propagation direction. In addition, a 32 μm diameter polyethylene particle was manipulated in the x-y plane by using the manufactured 4.32 MHz twin-trap device at 15  V pp (see supplementary material, Fig. S3 for further details).

FIG. 3.

Three-axis acoustic manipulation of a single 130 μm polyethylene particle using the 1.76 MHz device. Displacement in the x-axis direction (a), in the y-axis direction (b), and in the z-axis direction (c). Each of the images show the starting position of the particle as a dotted green circle, and the dotted white arrow indicates the direction of the movement through time. Multimedia available online.

FIG. 3.

Three-axis acoustic manipulation of a single 130 μm polyethylene particle using the 1.76 MHz device. Displacement in the x-axis direction (a), in the y-axis direction (b), and in the z-axis direction (c). Each of the images show the starting position of the particle as a dotted green circle, and the dotted white arrow indicates the direction of the movement through time. Multimedia available online.

Close modal

To find the applied force in the twin-trap, a force balance was performed against relative flow48 by moving the twin-trap in the x-direction relative to a stationary Petri dish. The drive voltage was adjusted until the radiation force was just able to hold a 130 μm polyethylene particle steady against the relative motion. At this point, the inertia can be neglected, and the acoustic radiation force equated to the drag force, F ARF = F s. As the velocities were low, Stokes' drag was assumed, hence F s = 6 π ν rU, where r is the radius of the particle, ν is the dynamic viscosity of water, and U = 300 μm/s is the steady relative velocity between the particle and the fluid.58 The voltage at which the trap was just able to hold the particle was measured to be 14.0 ± 0.2  V pp at which point, from the force balance, the radiation force in the x-direction was 36.7 nN. For the case of small dense/stiff particles, the observed 3D trapping can be explained by analyzing the pressure and particle velocity fields of the twin-trap with the Gor'kov equations. This analysis reveals that in the x-direction, the pressure gradients dominate, whereas in the y- and z-directions, the particle velocity gradients are also significant.

At a drive voltage of 14  V pp, hydrophone scans were performed using a fiber optic hydrophone (FOHS71, Precision Acoustics, UK) and the results are shown in Figs. 2(b) and 2(d), from which the pressure at the main twin-trap lobes was found to be 238.1 ± 21.4 kPa. The measurements were performed without the Petri dish and particles. See the supplementary material for further details on the hydrophone system and measurement.

To further explore the 3D manipulation capabilities of the 1.76 MHz twin-trap device, a 130 μm polyethylene particle was trapped and manipulated within a 2 mm external diameter, 0.5 mm thickness PTFE tube. The tube was placed on the visualization layer, and a particle was first positioned inside the tube using a syringe. The twin-trap device was activated at 45  V pp and positioned on the particle using the visualization layer to guide it into position. The twin-trap device was then used to move the particle along the tube and finally extract it, as shown in Fig. 4(a) (Multimedia view).

FIG. 4.

Example particle manipulation applications. (a) Manipulation of a 130 μm polyethylene particle inside a plastic tube. The time from the initial position to the final position is 0.3 s, with the green dotted circle showing the initial position of the particle. (b) Manipulation of a 920 μm diameter neuroectoderm aggregate. The total time from the initial to the final position is 18 s, with the green dotted circle showing the initial position of the aggregate. Note that between (a) and (b) the color of the mica particles in the visualization layer was changed to enhance the observation. Multimedia available online.

FIG. 4.

Example particle manipulation applications. (a) Manipulation of a 130 μm polyethylene particle inside a plastic tube. The time from the initial position to the final position is 0.3 s, with the green dotted circle showing the initial position of the particle. (b) Manipulation of a 920 μm diameter neuroectoderm aggregate. The total time from the initial to the final position is 18 s, with the green dotted circle showing the initial position of the aggregate. Note that between (a) and (b) the color of the mica particles in the visualization layer was changed to enhance the observation. Multimedia available online.

Close modal

To explore the twin-trap potential for biomedical applications, the manipulation of a 920 μm diameter 3D neuroectoderm aggregate was demonstrated, as shown in Fig. 4(b) (Multimedia view). The neuroectoderm aggregate was placed within a Petri dish including the visualization layer, and then, the 1.76 MHz twin-trap device was activated at 45  V pp and positioned on the cell aggregate using the visualization layer as a guide (see supplementary material for more information on the neuroectoderm aggregate). Once the aggregate was within the trap, it could be moved in the x-y plane as shown in Fig. 4(b). We note that the size of the neuroectoderm aggregate was comparable to the wavelength, hence well beyond the Rayleigh regime, and less than the spacing between the two high amplitude pressure lobes. This result suggests that in some cases particles of size comparable to the wavelength can be manipulated with this twin-trap device.

In this work, single beam acoustic twin-trap devices have been demonstrated that can manipulate a range of particle sizes between 32 and 920 μm. This included both inanimate polymer particles and fixed neuroectoderm aggregates. The twin-trap was shown to perform stable trapping and manipulation in all three spatial dimensions. The parts required for the construction of the twin-trap are simple and widely available as are the electronics needed to drive the device. To further facilitate ease of use, a visualization layer was developed to enable the location of the twin-trap to be monitored during manipulation. These acoustic manipulation capabilities offer potential for further applications in the biomedical sector, for example in tissue biofabrication and drug delivery.

See the supplementary material for additional figures of the experimental setup, finite element analysis on the performance of the printed lens, the manipulation of the 32 μm particle with the 4.32 MHz twin-trap device, a schematic to explain the mechanism of visualization, information on the preparation of the neuroectoderm aggregate, the fiber optic hydrophone (FOH) measuring system, and videos showing our experimental results.

The authors acknowledge Rob Hughes, Francisco Alvarez, Alexis Arroyo, Krishna Coimbatore Balram, and Xiaoyu Sun for their assistance in performing the experiments; Adrian Crimp and Paul Chappell for their support on the mechanical workshop; and WiCell and the provider scientist (James A Thomson, University of Wisconsin, Madison) as the original source of the WA09 (H9) human embryonic stem cell line, used at the University of Bristol for the research programme entitled “Symmetry-breaking technologies for cerebral organoid engineering.”

Mario E. Ortega-Sandoval was funded by CONAHCYT (Scholarship No. 795435). James P. K. Armstrong acknowledges funding from a UKRI Future Leaders Fellowship (No. MR/V024965/1).

The authors have no conflicts to disclose.

Mario E. Ortega Sandoval: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). Luke Cox: Formal analysis (supporting); Writing – review & editing (supporting). Amanda Franklin: Writing – review & editing (supporting). Martha Lavelle: Resources (supporting); Writing – review & editing (supporting). James P. K. Armstrong: Conceptualization (equal); Resources (equal); Supervision (equal); Writing – review & editing (equal). Bruce W. Drinkwater: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (lead); Software (lead); Supervision (lead); Validation (equal); Visualization (supporting); Writing – original draft (supporting); Writing – review & editing (equal).

The data that support the findings of this study are available within the article and its supplementary material.

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