ABSTRACT Contactless microscale tweezers are highly effective tools for manipulating, patterning, and assembling bioparticles. However, current tweezers are limited in their ability to

comprehensively manipulate bioparticles, providing only partial control over the six fundamental motions (three translational and three rotational motions). This study presents a joint

subarray acoustic tweezers platform that leverages acoustic radiation force and viscous torque to control the six fundamental motions of single bioparticles. This breakthrough is significant

as our manipulation mechanism allows for controlling the three translational and three rotational motions of single cells, as well as enabling complex manipulation that combines controlled

bioparticles with acoustic tweezers and taking images of each orientation using a standard microscope. With these capabilities, we anticipate the JSAT platform to play a pivotal role in

various applications, including 3D imaging, tissue engineering, disease diagnostics, and drug testing. SIMILAR CONTENT BEING VIEWED BY OTHERS MOVABLE SURFACE ACOUSTIC WAVE TWEEZERS: A

VERSATILE TOOLBOX FOR MICROMANIPULATION Article Open access 28 October 2024 ACOUSTIC BLACK HOLE EFFECT ENHANCED MICRO-MANIPULATOR Article Open access 12 October 2024 SPATIALLY SELECTIVE

MANIPULATION OF CELLS WITH SINGLE-BEAM ACOUSTICAL TWEEZERS Article Open access 25 August 2020 INTRODUCTION Precise single-cell manipulation, encompassing translation1,2, rotation3, and

deformation4,5, is essential for cellular biology, biophysics, and biomedical engineering. The ability to translate living cells in a contact-free manner significantly enhances the

capabilities of tissue engineering technologies to produce biomimetic tissues for numerous applications in regenerative medicine, and disease modeling2. Moreover, controllable rotational

manipulation enables high-resolution 3D reconstruction of cells3,6, and small organisms7, thereby unveiling hidden details pertaining to cellular structure and organization. This ability has

proven valuable in various areas, including cell profiling, disease diagnostics, and drug screening3. Furthermore, the ability to gradually deform a cell allows for the characterization of

cell mechanical properties, particularly those sensitive to changes in cytoskeletal and nuclear components, offering a label-free method for evaluating these alterations5. This ability is

also critical for mechanical phenotyping, cell classification, and the tracking of cellular metabolic dynamics8,9,10 with wide-ranging applications in biophysics and

biology11,12,13,14,15,16. Although various techniques have been developed to precisely manipulate cells, including optical tweezers17,18,19,20, optoelectronic tweezers21, magnetic

tweezers22,23, acoustic tweezers24,25,26,27,28,29, acousto-dielectric tweezers30, dielectrophoresis31,32,33, electrorotation3, and high-speed hydrodynamic flow14, the ability to manipulate

cells with complete control over all six fundamental motions remained a formidable challenge. Optical tweezers have gained popularity as a technique for manipulating micro/nano-particles18;

however, they require complex equipment and precise alignment of optical components while also introducing the risk of laser-induced heating that may cause physiological damage to

bioparticles. Optoelectronic tweezers offer the advantage of dynamically trapping cells with less power than traditional optical methods21. However, they fall short when attempting to

achieve in-plane cell rotation and deformation. Moreover, their usages are limited by the requirement of high ionic concentration environments due to ionic shielding and Joule heating21.

Magnetic tweezers are constrained by the complexity involved in embedding or attaching magnetic nanoparticles to cells, which may lead to cell damage34. While dielectrophoresis33,

electrorotation3, and high-speed hydrodynamic manipulation14,35 can accomplish cell translation, rotation, or deformation, respectively, none of these techniques possess all of the

aforementioned functions. In addition, they lack the ability to achieve and control all six fundamental motions. In recent years, acoustofluidics has emerged as a promising contact-free

strategy for cell manipulation, offering the advantages of label-free operation and excellent biocompatibility36,37,38,39,40,41,42,43. With these features, acoustofluidic technologies have

found numerous applications in bioparticle separation44,45,46,47,48,49, exosome enrichment50,51,52, and cell patterning53,54,55, among others56,57,58,59,60,61,62. These applications

typically leverage the acoustic radiation force that results from bioparticle-induced acoustic field changes63,64,65,66,67, as well as the acoustic streaming that stems from acoustic energy

dissipation in fluids68,69,70. Acoustic tweezers based on orthogonally arranged two pairs of transducers with phase and amplitude modulation capabilities enable 3D translation of single

cells2. For rotational object manipulation, an early study by Busse and Wang presented a theoretical framework to predict the torque induced by orthogonal acoustic waves71. To achieve both

translational and rotational manipulation of acoustically trapped objects, Marzo and Drinkwater developed holographic acoustic tweezers, leveraging an array of transducers to generate

airborne acoustic waves and reshape the acoustic energy field to versatile patterns72. However, as this method uses airborne acoustic waves, it is limited to manipulating objects in the

air72. In addition to translational and rotational object manipulation, bulk acoustic wave- and streaming-based approaches have been developed to deform cells6,15,73,74,75,76. Given the

successes of previous studies, in the context of single-cell manipulation using surface acoustic waves (SAWs), no SAW device achieves the three critical cell manipulation functions:

controllable translation, rotation, and deformation of cells. Moreover, few studies investigate the mechanisms to achieve complex manipulation that combines controlled translation and

rotation of single cells. This study presents an acoustic tweezer platform, termed the joint subarray acoustic tweezers (JSAT) system, which allows for controlling the six fundamental (three

translational and three rotational) motions of single cells, achieving complex motions with controlled translation and rotation, and deforming an acoustically trapped cell, by leveraging

SAW-induced radiation force and acoustic streaming vortex-induced shear force. By tuning the phases and amplitudes of orthogonal standing SAWs at low frequencies, the JSAT system traps cells

and controls the trapped cell’s three translational motions (i.e., _u__x_, _u__y_, and _u__z_). To control the cell’s rotational motions (i.e., _θ__x_ and _θ__y_), high-frequency standing

SAW-induced acoustic streaming vortices are utilized to apply viscous torques on the trapped cell. Similarly, acoustic streaming vortices77,78 induced by high-frequency traveling SAWs

control the trapped cell’s rotational degree-of-freedom (DoF) _θ__z_. Unlike existing acoustic tweezers, our JSAT system is able to control all six fundamental motions and achieve complex

manipulation combined with controlled translational and rotational motions, thus facilitating comprehensive cell manipulation in a 3D space. In addition to these features, our system can

control the force applied to an acoustically trapped cell to gradually deform the cell. This ability allows for the introduction of controllable mechanical perturbations to single cells for

detailed characterization of cellular responses at different levels of mechanical perturbations. RESULTS MECHANISMS OF JSAT The JSAT system can exert and control both the acoustic radiation

force and viscous torque applied on a cell to enable precise and controllable six fundamental motions (three translational and three rotational motions) manipulation of the cell. To enable

this ability, our JSAT system leverages a unique array of interdigital transducers (IDTs) deposited on a LiNbO3 substrate to generate and control both the SAW and acoustic streaming fields

inside a polydimethylsiloxane (PDMS)-based microfluidic chamber. As illustrated by a device schematic in Fig. 1a (also see Supplementary Fig. S1), the entire array is composed of three

(i.e., inner, middle, and outer) subarrays that have collections of 4, 4, and 8 IDTs (denoted as {\({{\mbox{IDT}}}_{{{\mathrm{i}}}}^{{{\mathrm{out}}}}\)}4,

{\({{\mbox{IDT}}}_{{{\mathrm{j}}}}^{{{\mathrm{mid}}}}\)}4, and {\({{\mbox{IDT}}}_{{{\mathrm{k}}}}^{{{\mathrm{in}}}}\)}4, respectively) for generating SAWs at different frequencies. Such

design is difficult to achieve using thickness-mode piezoelectric transducers (such as PZTs), as the bulk acoustic waves generated by a PZT are impeded by other PZTs placed in the wave

propagation path. This limits the options of integrating multiple PZTs for achieving complex cell/particle manipulation functions. Conversely, SAWs generated by an IDT exhibit good

transmissibility through regions with IDTs working at different frequencies, thereby allowing more options for designing complex IDT arrays. Supplementary Fig. S2 shows a photo of a

fabricated JSAT device with an IDT array and a microfluidic chamber. The mechanisms for achieving multi-DoF manipulation of single cells using the three subarrays are presented below. The

outer subarray enables cell trapping by leveraging acoustic radiation force and controlling the trapped cell’s three translational motions (i.e., _u__x_, _u__y_, and _u__z_) through phase

and amplitude modulations of SAWs. This subarray has two orthogonal pairs of IDTs positioned along the _x_- and _y_-axes, respectively. When applying excitation signals to these IDTs at

their resonance frequencies, a grid-like standing SAW field is formed within the microfluidic chamber. Consequently, a cell can be trapped by one of multiple Gor’kov potential wells due to

the acoustic radiation force79,80 applied to the cell. Moreover, the trapped cell can be precisely translated with a displacement of _u__x_ (or _u__y_), by moving the Gor’kov potential

well’s position through phase modulation, i.e., changing the phase difference between the two IDTs along the _x_- (or _y_-) axis. In addition, the SAW energy leaked into the fluid domain in

the PDMS chamber induces an out-of-plane acoustic radiation force, as illustrated in Fig. 1b (top right). The out-of-plane translation _u__z_ of the acoustically trapped cell can be

precisely controlled by adjusting the IDT excitation voltage, which regulates the force component responsible for the translation. The middle subarray controls the trapped cell’s rotational

DoFs _θ__x_ and _θ__y_ by utilizing standing SAW-induced acoustic streaming vortices. The middle subarray’s two IDTs positioned along the _x_-axis generate acoustic streaming vortices with

non-zero angular momentums, enabling precise control of cell rotation _θ__x_, as shown in Fig. 1b (bottom left). Similarly, the two IDTs along the _y_-axis can generate vortices, enabling

precise control of cell rotation _θ__y_, as illustrated in Fig. 1b (bottom middle). The operation frequencies of these middle subarray IDTs are higher than the frequencies of the outer

subarray IDTs, so the diameter (~ 25 µm) of each streaming vortex is comparable to the cell size. The generated streaming vortices can apply a viscous torque to the cell, introducing angular

momentum. Moreover, the cell angular velocities can be altered by adjusting the input voltages applied to the middle subarray. The inner subarray controls the trapped cell’s rotation _θ__z_

by manipulating the traveling SAW-induced acoustic streaming vortices. Here, the inner subarray’s four IDTs with even subscripts (see Supplementary Fig. S1) are utilized to generate

traveling SAWs whose energy beam directions are tangential to a circle. These traveling SAWs can induce an in-plane acoustic streaming vortex with a clockwise angular momentum, resulting in

the ability to rotate a cell clockwise. On the other hand, the four IDTs with odd subscripts can be used to generate a counterclockwise acoustic streaming vortex to enable the

counterclockwise rotation of a cell, as illustrated in Fig. 1b (bottom right). Moreover, by tuning the input voltages for these IDTs, the angular movement of the generated streaming vortex

can be adjusted to control the angular velocity of the cell. As presented above, by leveraging three subarrays of IDTs, our JSAT system can trap a cell and control the cell’s six fundamental

motions. Moreover, through the superposition of forces/torques generated by IDTs to control different DoFs, our JSAT system enables complex manipulation of a trapped cell, allowing for

simultaneous translation and rotation. Furthermore, by slowly increasing the force applied on a cell, our device can gradually deform an acoustically trapped cell. We have performed

simulations and experiments with key results reported below to achieve and validate these functions. 3D TRANSLATION VIA JSAT To trap a cell and control its 3D translational motion, the outer

subarray leverages two orthogonal pairs of IDTs (see Fig. 2a) to generate SAWs with the same wavelength (λout = 200 μm) in both the _x-_ and _y-_ directions. The interaction of SAWs

generated from these four IDTs leads to a square grid-like distribution of pressure nodes (or antinodes) with the same period of λout/2 along the _x-_ and _y-_ directions. At each pressure

node, the resulting Gor’kov potential well can trap a cell with a positive acoustic contrast factor81. Since cells typically have dimensions in tens of microns, the low frequencies used

ensure the in-plane Stokes drag force resulting from acoustic streaming is negligible compared to the in-plane acoustic radiation force for trapping the cell. To achieve controllable

in-plane translations _u__x_ and _u__y_ of a trapped cell, our system leverages the phased modulation approach, which tunes the phase differences _φ__x_ and _φ__y_ between excitation signals

for the two IDTs along the _x-_ and _y-_directions, respectively. As shown by the simulated acoustic energy fields, when phase differences change from _Φ_ (Fig. 2b, left) to _Φ_ + Δ_Φ_

(Fig. 2b, right), where _Φ_ = [_φ__x_, _φ__y_] and Δ_Φ_ = [Δ_φ__x_, Δ_φ__y_], the potential well with the trapped cell can be shifted by a short translation vector _U_ = [_u__x_, _u__y_, 0]

with _u__x_ = Δ_φ__x_λout/(4π) and _u__y_ = Δ_φ__y_λout/(4π). Based on this mechanism, a long and complex translation path can be achieved by discretizing the path into a series of short

translation vectors, calculating the required phase adjustments for these vectors, and sequentially applying the phase adjustments to IDT excitation signals. To execute this translation

process automatically, the phase adjustments can be sequentially applied through two dual-channel function generators controlled by MATLAB code. To demonstrate this ability, we successfully

guided an MCF7 cell’s movement to depict the letters ‘D’, ‘U’, ‘K’, and ‘E’ (see Supplementary Movie 1). The actual cell trajectories (see Fig. 2c) were revealed by stacking the microscopic

images captured during the dynamic cell translation process. As shown in Supplementary Fig. S3, the actual cell positions closely agree with the predicted positions using relations _u__x_ = 

Δ_φ__x_λout/(4π) and _u__y_ = Δ_φ__y_λout/(4π). As the phase modulation-based translational manipulation mechanism is known for its good predictability2, we didn’t perform any calibration

before translating an MCF7 cell following complex trajectories. To achieve controllable out-of-plane translation _u__z_ of a trapped cell, our system leverages the amplitude modulation

approach, which adjusts the excitation amplitudes of the outer subarray’s four IDTs. The experimental results demonstrate that the position of an MCF7 cell can be shifted in the + 

_z_-direction through acoustic waves generated by the IDTs (see Fig. 2e and Supplementary Movie 2). To better elucidate the manipulation mechanism, finite element simulations were performed.

As SAWs propagate in the LiNbO3 substrate, their energy leaks into the fluid domain above the substrate, leading to an energy flux along the Rayleigh angle direction, as illustrated in Fig.

 2d. Therefore, a cell within the vicinity is subjected to an out-of-plane acoustic radiation force component \({{{\mathrm{F}}}}_{{{{\mathrm{rad}}}}\_{{{\mathrm{z}}}}}^{{{\mathrm{out}}}}\),

which points to the + _z_-direction acting as the levitation driving force, as predicted by simulation results in Supplementary Fig. S4. Additional theoretical investigation of the

out-of-plane acoustic radiation force can be found in our previous article82. An acoustofluidic simulation was also performed to analyze the acoustic streaming-induced out-of-plane Stokes

drag force. As shown in the simulation results (Supplementary Fig. S5 and Fig. 2d, middle), there is a downward acoustic streaming at the pressure node, thus inducing a – _z_-direction drag

force that gradually diminishes from the microfluidic chamber’s center to the bottom and top (see Supplementary Fig. S5). The out-of-plane translation _u__z_ depends on the interplay of all

the out-of-plane forces, including the position-dependent acoustic radiation and drag forces, the buoyancy force FBuo, and the gravitational force Fg, as shown in Fig. 2d (right). Most

cells, including MCF7 cells, have a density slightly higher than the culture medium (water with additives), so the _z_-directional manipulation can be controlled theoretically by applying a

precise input power to match to account for the aforementioned out-of-plane forces. However, the control of out-of-plane translation cannot be as precise and stable as the control of

in-plane translation due to the absence of a Gor’kov potential well-like trap. In addition, when the cell experiences other motions, especially rotational motions, they affect the

out-of-plane translation. The out-of-plane translation precision is also affected by the two-dimensional imaging nature of our current microscope, as the translation is difficult to be

quantitively monitored. 3D ROTATION VIA JSAT To control an acoustically trapped cell’s 3D rotational motion, our JSAT device leverages the middle and inner subarrays of IDTs to generate and

control acoustic streaming vortices for applying and controlling viscous torques on the cell. The \({{{\mathrm{IDT}}}}_{2}^{{{\mathrm{mid}}}}\) and

\({{{\mathrm{IDT}}}}_{4}^{{{\mathrm{mid}}}}\) of the middle subarray are for controlling the cell rotation _θ__x_ using acoustic streaming, as illustrated in Fig. 3a. They generate standing

SAWs with a wavelength _λ_mid of 100 μm, further inducing tunnel-like streaming vortices with counter chiralities at different sides of each pressure node, as revealed by the results in

Supplementary Fig. S6 and Supplementary Movie 3. Therefore, for a cell trapped at a pressure node, the two counter-chirality vortices both apply torques to the cell, and the competition

between the two vortices determines the cell’s angular motion. To better elucidate the cell manipulation mechanism, we performed numerical simulations with the excitation voltage

\({{{{\rm{V}}}}}_{2}^{{{{\mathrm{mid}}}}}\) for \({{{\mathrm{IDT}}}}_{2}^{{{\mathrm{mid}}}}\) slightly higher than the voltage \({{{{\rm{V}}}}}_{4}^{{{{\rm{mid}}}}}\) for

\({{{\mathrm{IDT}}}}_{4}^{{{\mathrm{mid}}}}\). As shown in Supplementary Fig. S6, the streaming fields of vortices on different sides of a pressure node become slightly asymmetric. These

asymmetric vortices can lead to a non-zero viscous torque applied on the cell, consequently inducing cell rotation. Moreover, our simulation results (Supplementary Fig. S6d, S6e) reveal that

the tangential streaming velocities can lead to cell rotation in the + _θ__x_-direction being dominant. Furthermore, during the streaming-induced cell rotation, the acoustic potential well

generated by the standing SAWs can still effectively trap the cell, ensuring no translational motion or eccentricity. The experimental validation results (top of Fig. 3b and Supplementary

Movie 3) show that an MCF7 cell could be successfully trapped and rotated in the + _θ__x_-direction when \({{{{\rm{V}}}}}_{4}^{{{{\rm{mid}}}}}\)<\({{{{\rm{V}}}}}_{2}^{{{{\rm{mid}}}}}\),

agreeing with the numerically predicted rotation direction. Therefore, we can reliably execute the rotational manipulation as planned. Similarly, under the condition of

\({{{{\rm{V}}}}}_{4}^{{{{\rm{mid}}}}}\)>\({{{{\rm{V}}}}}_{2}^{{{{\rm{mid}}}}}\), an acoustically trapped MCF7 cell exhibited rotation in the – _θ__x_-direction (Fig. 3b, bottom and

Supplementary Movie 3). Note that the aforementioned rotation control approach is for a cell located near the center of the microfluidic chamber, as the SAWs emitted from a pair of IDTs

travel to the center region with similar attenuation lengths. When the cell position is close to either \({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\) or

\({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\), asymmetric streaming vortices can still be generated at different sides of a pressure node even

\({{{{\rm{V}}}}}_{2}^{{{{\rm{mid}}}}}\)=\({{{{\rm{V}}}}}_{4}^{{{{\rm{mid}}}}}\), enabling cell rotation in the ± _θ__x_-directions. To investigate the relationship between input voltage and

cell rotation speed, we loaded an MCF7 cell at a distance of _λ_mid/2 away from the center, followed by applying the same voltage to \({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\) and

\({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\). As depicted in Fig. 3c, the rotation speed exhibited a positive correlation with the input voltage and could reach values as high as ~ 450 RPM in

the cell culture medium. Furthermore, the cell rotation revolution shows a nearly linear relationship with time (see Supplementary Fig. S7). These findings indicate that the cell’s spinning

rate remains relatively stable during the streaming generation period. In addition, by activating \({{{{\rm{IDT}}}}}_{1}^{{{{\rm{mid}}}}}\) and \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{mid}}}}}\) to

generating streaming vortices, we successfully induced the rotation of an MCF7 cell in the ±_θ__y_-directions (Fig. 3e and Supplementary Movie 4). The corresponding relationship between

input voltage and rotation speed is summarized in Fig. 3f. For enabling cell rotation in the _θ__z_-direction, the inner subarray (Fig. 3g) is used. When signals at resonant frequencies are

applied to the four IDTs with odd subscripts, the traveling SAW from each IDT produces a volume force that propels the fluid away from the IDT. Consequently, the combined effect of the four

driving flows forms a counterclockwise streaming vortex. Note that when only activating \({{{{\rm{IDT}}}}}_{1}^{{{{\rm{in}}}}}\) and \({{{{\rm{IDT}}}}}_{5}^{{{{\rm{in}}}}}\), a

counterclockwise streaming vortex can still be generated, as proven by numerical and experimental results (Supplementary Fig. S8 and Supplementary Movie 5). Therefore, an MCF7 cell, driven

by the viscous torque induced by the counterclockwise streaming vortex, can be rotated in the + _θ__z_-direction (see Fig. 3h, top and Supplementary Movie 5). Similarly, the IDTs with even

subscripts in the inner subarray can induce cell rotation in the – _θ__z_-direction. Through experiments with \({{{{\rm{IDT}}}}}_{2}^{{{{\rm{in}}}}}\) and

\({{{{\rm{IDT}}}}}_{6}^{{{{\rm{in}}}}}\) turned on, the results (Fig. 3h, bottom and Supplementary Movie 5) successfully confirm the induced cell rotation in the – _θ__z_-direction.

Furthermore, we experimentally investigated the relationship between input voltage and rotation speed. The findings reveal that the rotation speed exhibited an increasing trend as the input

voltage increased (Fig. 3i). The slight spin rate drops at 10 Vpp could be induced by measurement error. TUNABLE ROTATION VIA JSAT Our device can steer the cell rotation axis by

simultaneously activating the two orthogonal pairs of IDTs in the middle subarray for rotating a cell in the _θ__x_- and _θ__y_- directions (Fig. 3j). Supplementary Fig. S9a presents the

numerical results of acoustic streaming when _x_- and _y_-axes standing SAWs have the same amplitude. In the vicinity of a pressure node, the results show four vortices with angular momentum

vectors in − 45°, 45°, 135°, and − 135° directions, respectively. Similar to controlling the cell rotation in the _θ__x_-direction, the middle subarray can rotate a trapped cell in the same

direction as any one of those four vortices by adjusting the voltages applied to the four IDTs or placing the cell off-center. For instance, an MCF7 cell trapped at _x_ = _λ_mid/2 and _y_ =

 _λ_mid/2 was successfully spun to carry an angular momentum in the direction of − 45° (see Fig. 3k and Supplementary Movie 6), _x_- and _y_-axes SAWs had similar amplitudes. Moreover, by

carefully changing the amplitude ratio between _x_- and _y_-axes SAWs, the axis of cell rotation can be gradually steered. For example, when the ratio is 0.57:1, the simulation

(Supplementary Fig. S9b) predicts four vortices surrounding a pressure node carrying angular momentums in − 30°, 30°, 150°, and − 150°. The experimental results (bottom row of Fig. 4k and

Supplementary Movie 6) show a rotating MCF7 cell with respect to an axis at − 29°, close to the predicted direction of − 30°. When the SAW amplitude ratio further changes to 0.3:1, numerical

simulations in Supplementary Fig. S9c reveal the four vortices with angular momentums at − 15°, 15°, 165°, and − 165°. The experimental results (Supplementary Fig. S9d and Supplementary

Movie 6) confirm that an MCF7 cell trapped at _x_ = _λ_mid/2 and _y_ = _λ_mid/2 can be rotated to carry an angular momentum in the direction of − 15°, agreeing with the numerical prediction.

Note that when adjusting input voltages for IDTs, the different SAW generation efficiencies in the _x_- and _y_- directions of the used Y128-cut LiNbO3 wafer must be considered.

SIMULTANEOUS TRANSLATION AND ROTATION VIA JSAT A unique feature of our JSAT platform is its ability to perform simultaneous cell translation and rotation. To understand such compound motion,

we conducted simulations and experiments involving different combinations of translation and rotation directions. Figure 4a (left) illustrates the mechanism to simultaneously translate a

trapped cell in the _x_-direction and rotate the cell in the _θ__x_-direction. The translation is controlled by tuning the input phase difference between

\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\) and \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\), and the rotation is controlled by the streaming vortex generated by

\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\) and \({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\). Supplementary Fig. S10 compares acoustic energy distributions of standing SAWs generated by one

transducer pair {\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\), \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\)} and two transducer pairs {\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\),

\({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\)} and {\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\), \({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\)}. The _x_ position with minimum energy is unchanged upon

introducing the _y_-axis SAWs from {\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\), \({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\)}, meaning that the positional stability of a trapped cell is

unaffected by the _y_-axis SAWs. On the other hand, we found that the _x_-axis standing SAW exhibits negligible impact on the generation of streaming vortices with angular momentum vectors

in ± _x_-directions. These simulation results support the simultaneous execution of cell translation and rotation and control these motions using transducer pairs

{\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\), \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\)} and {\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\), \({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\)},

respectively. For the proof-of-concept, we successfully utilized these two transducer pairs to achieve controlled translation in the _x_-direction and rotation in the _θ__x_-direction

(Supplementary Movie 7 and Fig. 4a, right). As the _x_-axis standing SAW generated from {\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\), \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\)} exhibits

negligible impact on the streaming vortices generated by {\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\), \({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\)} and the two streaming vortex tunnels are

parallel to the cell translation direction, our approach achieves continuous cell rotation during the translation process. Similarly, we manipulated an MCF7 cell to undergo both translation

in the _y_-direction and rotation in the _θ__y_-direction (Supplementary Movie 7 and Fig. 4b, right) by using transducer pairs {\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{out}}}}}\)

\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{out}}}}}\), \({{{{\rm{IDT}}}}}_{4}^{{{{\rm{out}}}}}\)} and {\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{mid}}}}}\), \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{mid}}}}}\)}. The

mechanism to enable simultaneous cell translation in the _x_-direction and rotation in the _θ__y_-direction is illustrated in Fig. 4c (left). Here, the translation control is achieved by

tuning the phase difference between inputs for \({{{{\rm{IDT}}}}}_{1}^{{{{\rm{mid}}}}}\) and \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{mid}}}}}\), and the rotation is enabled by streaming vortices

generated by the _x_-axis standing SAWs. To demonstrate this approach, we performed an experiment with \({{{{\rm{IDT}}}}}_{1}^{{{{\rm{mid}}}}}\) and \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{mid}}}}}\)

turned it on. By gradually adjusting the phase difference, our results (Supplementary Movie 8 and Fig. 4c, right) show that the generated standing SAWs can translate an MCF7 cell in the

_x-_direction while inducing acoustic streaming to spin the cell in the _θ__y_-directions. Although both acoustic radiation force and streaming are generated by the same IDTs, the

translation _u__x_ is controlled by the input phase difference, and the rotation _θ__y_ is controlled by the input amplitude difference between the two IDTs. Hence, the involved

translational and rotational motions could be independently controlled. Moreover, when translating a cell through phase modulation, the positions of two streaming vortex tunnels (illustrated

in Fig. 4c) shift synchronously, thus ensuring continuous cell rotation during the translation process. By using the above mechanisms with \({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\) and

\({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\), we achieved simultaneous translation of an MCF7 in the _y-_direction and rotation in the _θ__x_-direction (Fig. 4d and Supplementary Movie 8). The

mechanism to achieve simultaneous cell translation in the _x_-direction and rotation in the _θ__z_-direction is illustrated in Fig. 4e (left). By tuning the phase difference between inputs

for \({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\) and \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\), the pressure node position of an _x_-axis standing SAW can be adjusted to translate a trapped

cell in the _x_-direction. On the other hand, the _θ__z_-direction cell rotation can be achieved using the streaming vortex induced by the traveling SAWs from

\({{{{\rm{IDT}}}}}_{3}^{{{{\rm{in}}}}}\) and \({{{{\rm{IDT}}}}}_{7}^{{{{\rm{in}}}}}\). By comparing the simulated acoustic energy fields (Supplementary Fig. S11) generated by one transducer

pair {\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\), \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\)} and two transducer pairs {\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\),

\({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\)} and {\({{{{\rm{IDT}}}}}_{3}^{{{{\rm{in}}}}}\), \({{{{\rm{IDT}}}}}_{7}^{{{{\rm{in}}}}}\)}, the acoustic energy field experiences small changes when

considering the traveling SAWs from {\({{{{\rm{IDT}}}}}_{3}^{{{{\rm{in}}}}}\), \({{{{\rm{IDT}}}}}_{7}^{{{{\rm{in}}}}}\)}. On the other hand, the simulated streaming field (Supplementary Fig.

 S11) shows a counterclockwise streaming vortex. These simulation results support the simultaneous control of the cell translation in the _x_-direction and the rotation in the

_θ__z_-direction by leveraging transducer pairs {\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{out}}}}}\), \({{{{\rm{IDT}}}}}_{3}^{{{{\rm{out}}}}}\)} and {\({{{{\rm{IDT}}}}}_{3}^{{{{\rm{in}}}}}\),

\({{{{\rm{IDT}}}}}_{7}^{{{{\rm{in}}}}}\)}, respectively. Our experiment successfully achieved simultaneous translation of an MCF7 cell in the _x_-direction and rotation in the

_θ__z_-direction (Supplementary Movie 9 and Fig. 4e, right). Similarly, we achieved concurrent translation and rotation of an MCF7 cell in the _y_-and _θ__z_-directions (Supplementary Movie 

9 and Fig. 4f, right) by using transducer pairs {\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{out}}}}}\), \({{{{\rm{IDT}}}}}_{4}^{{{{\rm{out}}}}}\)} and {\({{{{\rm{IDT}}}}}_{1}^{{{{\rm{in}}}}}\),

\({{{{\rm{IDT}}}}}_{5}^{{{{\rm{in}}}}}\)}. DEFORMING SINGLE CELLS VIA JSAT Our JSAT platform can locally deform single cells and control cell motion. As illustrated in Fig. 5a, when an

acoustically trapped cell is positioned within a high angular momentum region of streaming vortices induced by standing SAWs generated by {\({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\),

\({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\)}, the streaming velocity in the cell’s viscous boundary layer drops rapidly to the same value as the cell’s surface velocity, thereby subjecting the

cell to a large shear stress. Notably, the cell’s surface velocity and shear stress exhibit a decreasing trend from the equator to the poles, prompting cell elongation along the axis of

rotation (i.e., _x_-axis). The acoustic radiation force induced by _y_-axis standing SAWs also contributes to cell elongation. To experimentally deform an MCF7 cell, we applied 10 Vpp

excitation signals to \({{{{\rm{IDT}}}}}_{2}^{{{{\rm{mid}}}}}\) and \({{{{\rm{IDT}}}}}_{4}^{{{{\rm{mid}}}}}\). The obtained time-sequential images (Fig. 5b) and Supplementary Movie 10

clearly show the dynamic process of gradually deforming a trapped cell over time. We also conducted a quantitative analysis by evaluating the gradual cell deformation over time and

investigating the effect of the IDT excitation voltage on cell deformation. At 6 Vpp, the shape of an MCF7 cell remains almost unchanged throughout the 100-second observation period (see

Fig. 5c). At a higher voltage of 8 Vpp, the cell deformation data exhibits a nearly linear trend with time. When the input voltage was further increased to 10 Vpp, the cell deformation

initially experienced a quick increase, followed by a slight decrease, and then converged to a relatively stable value. These findings indicate that the time-dependent cell deformation

processes demonstrate distinct trends under different IDT excitation voltages, highlighting our JSAT device’s ability to controllably deform acoustically trapped cells by tuning the

generated SAWs and streaming. DISCUSSION In this study, we successfully developed and demonstrated a JSAT platform that is capable of controlling the six fundamental motions of single cells,

including three translational (i.e., _u__x_, _u__y_, and _u__z_) and three rotational (i.e., _θ__x_, _θ__y_, and _θ__z_) components. Our unique IDT array, composed of an outer, middle, and

inner subarray, enables this ability. The in-plane translations (_u__x_ and _u__y_) of a cell trapped in a Gor’kov potential well are achieved by shifting the potential well’s position and

tuning input signal phases for the outer subarray. By adjusting the input signal voltages for the outer subarray, our JSAT platform allows for controlling the out-of-plane translation

(_u__z_), i.e., controlling a cell’s _z_ position with acoustic radiation, buoyancy, drag, and gravitational forces. The controllable rotations (_θ__x_ and _θ__y_) of an acoustically trapped

cell are accomplished based on acoustic streaming vortices induced by high-frequency standing SAWs from the middle subarray, and the rotation _θ__z_ is achieved by using acoustic streaming

vortices generated by multiple traveling SAWs, whose energy beam directions are all tangential to a circle. With the aforementioned features, such as achieving the six fundamental motions of

single cells and locally deforming a cell, our JSAT platform represents a significant advancement compared to previous technologies, such as optical, magnetic, and acoustic tweezers. Our

experiments successfully validated the ability to independently control the multi-DoF motions of an acoustically trapped cell, while none of the previously developed tweezers could provide

this ability. Moreover, our experiments demonstrated the ability to realize complex combined motions, such as _u__x_ and _θ__x_, _u__x_ and _θ__z_, _u__y_ and _θ__z_, etc., while previous

tweezers could not perform these complex manipulations. Furthermore, our JSAT platform can gradually deform an acoustically trapped cell without directly touching the cell by controlling the

applied shear force induced by streaming and acoustic radiation force from standing SAWs. Recording the dynamic cell deformation process makes it possible to characterize cell mechanical

properties83. Therefore, contactless tweezers that can gradually deform a cell hold significant potential for cell phenotyping, disease diagnosis, and drug testing applications. Beyond the

experiments conducted in this study, we will further test the ability to simultaneously deform multiple cells trapped in an array of potential wells. We also plan to introduce a feedback

control loop that integrates live imaging, real-time decision-making, and programmable input signal modulation to enable platform automation. In addition, beyond manipulating cells, we

expect that our tweezing mechanisms can be used for manipulating other bioparticles, such as embryos, bacteria, and extracellular vesicles, and these manipulations will be tested in our

future studies. In the long run, we anticipate that the JSAT platform can become a widely used tool facilitating various applications such as 3D cell imaging, single-cell analysis, tissue

engineering, disease diagnostics, and mechanobiology. METHODS DEVICE FABRICATION A schematic and a photo of the fabricated JSAT device are shown in Supplementary Figs. S1 and S2,

respectively. The JSAT device is composed of a PDMS microfluidic chamber and an array of IDTs on a Y128-cut LiNbO3 wafer (500 mm thick). To fabricate IDTs, we transferred the designed

electrode patterns to a wafer by standard photolithography84. Then, we conducted e-beam evaporation of 10 nm Cr and then 100 nm Au. After a lift-off process, we obtained a wafer with an IDT

array composed of outer, middle, and inner subarrays, whose electrode widths were 50 μm, 25 μm, and 10 μm, respectively. On the other hand, the PDMS micro-chamber was fabricated by using

standard soft-lithography and mold-replica steps. First, a 60 μm layer of photoresist (SU-8 50, KAYAKU) was coated on a 4-inch silicon wafer, followed by soft lithography and application of

an SU-8 Developer (KAYAKU). Then, PDMS was poured over the SU-8 mold, degassed, and cured at 65 °C for 1 h. The cured PDMS was peeled off the silicon wafer and cut into the desired

small-size blocks containing microfluidic chambers. The inlet and outlet of a microfluidic chamber were created with a puncher. The PDMS chamber and the SAW substrate with IDTs were surface

treated by oxygen plasma for 5 mins (Plasma Cleaner Atto, Diener Electronic) and then bonded, followed by a heat treatment at 65 °C for 24 h. PARTICLE AND CELL SAMPLE PREPARATION Polystyrene

particles (PSF-200NM, Magsphere) were suspended in deionized water to a concentration of ~ 1 × 106 cells/mL. MCF7 cells (ATCC) were cultured in DMEM (Gibco, Life Technologies), which

contains 10% fetal bovine serum (Gibco, Life Technologies) and 1% penicillin-streptomycin (Mediatech) in an incubator (Heracell Vios 160i CO2 incubator, Thermo Scientific) with a temperature

of 37 °C and a CO2 level of 5%. Before each experiment, cells were detached from the culture dish with trypsin-EDTA (Gibco, Life Technology) and resuspended in DMEM to a concentration of ~ 

1 × 106 cells/mL. DEVICE OPERATION To perform cell manipulation experiments using our fabricated JSAT device, the device was mounted on the sample stage of an inverted optical microscope

(TE2000U, Nikon). MCF7 cells suspended in DMEM were injected into the microfluidic chamber using a 1-mL syringe (309659, Becton Dickinson). To generate SAWs for applying acoustic radiation

and viscous shear forces on cells, the input signals were generated by two dual-channel arbitrary function generators (AFG3102C, Tektronix) and then directly sent to IDTs. Note that the

function generators were controlled by a MATLAB program to achieve automatic manipulation of acoustic and flow fields, along with the desired translation and rotation of an acoustically

trapped MCF7 cell in the microfluidic chamber. To translate a cell along the desired complex paths, the frequencies and voltages for the IDTs remain constant, while shifts are made to the

input phases. The phase shifts are automatically performed using customized MATLAB codes, having key features including adjusting the phases, frequencies, and amplitudes of multiple signal

channels, as well as gradually changing the phases following predetermined sequences of phases for achieving step-by-step translation of a cell. Images and videos were taken by a

charge-coupled device camera (CoolSNAP HQ2, Photometrics). The videos for cell rotation speed measurement were recorded by a high-speed camera (Fastcam SA4, Photron). NUMERICAL SIMULATIONS

The details of numerical simulation methods were provided in Supplementary Notes 1 and 2. CELL DEFORMATION MEASUREMENT To evaluate cell deformation from an acquired microscopic image, we

used feature and edge detection codes in Matlab to get the cell shape from the image. Then, the cell’s projected area _A_ and perimeter _l_ were extracted from the identified cell shape. By

further using a relation \(1-2\sqrt{\pi A}/l\), the cell deformation induced by our JSAT platform was elevated85. In addition, we measured the SAW device’s temperature, as well as the

post-treatment cell viability. Their detailed procedures and results are in Supplementary Note 3 and Supplementary Figs. 13–15. REPORTING SUMMARY Further information on research design is

available in the Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY The authors declare that all data and code supporting the findings of this study are available

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(2015). Article  PubMed  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We acknowledge the support from the National Science Foundation (CMMI-2104526 (Z.T.) and CMMI-2243771

(Z.T.)), the National Science Foundation Graduate Research Fellowship Program (2139754 (J.R.)), and the National Institutes of Health (R01GM144417 (Z.T.)). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC, USA Liang Shen, Kaichun Yang, Jianping Xia, Neil Upreti, Jinxin Zhang, Chuyi Chen, 

Nanjing Hao, Zhichao Pei & Tony Jun Huang * Department of Mechanical Engineering, Virginia Polytechnical Institute and State University, Blacksburg, VA, USA Liang Shen & Zhenhua Tian

* Department of Biomedical Engineering, Duke University, Durham, NC, USA Joseph Rich Authors * Liang Shen View author publications You can also search for this author inPubMed Google

Scholar * Zhenhua Tian View author publications You can also search for this author inPubMed Google Scholar * Kaichun Yang View author publications You can also search for this author

inPubMed Google Scholar * Joseph Rich View author publications You can also search for this author inPubMed Google Scholar * Jianping Xia View author publications You can also search for

this author inPubMed Google Scholar * Neil Upreti View author publications You can also search for this author inPubMed Google Scholar * Jinxin Zhang View author publications You can also

search for this author inPubMed Google Scholar * Chuyi Chen View author publications You can also search for this author inPubMed Google Scholar * Nanjing Hao View author publications You

can also search for this author inPubMed Google Scholar * Zhichao Pei View author publications You can also search for this author inPubMed Google Scholar * Tony Jun Huang View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS L.S. conceived the idea. L.S. developed the models for simulations. L.S. designed, fabricated, and

characterized JSAT devices. K.Y. and J.Z. fabricated the JSAT devices. L.S., N.H., Z.P., J.X., and C.C. conducted the experiments. L.S. analyzed the data. L.S., Z.T., J.R., N.U., and T.J.H.

wrote the paper. T.J.H. and Z.T. supervised the study. CORRESPONDING AUTHORS Correspondence to Zhenhua Tian or Tony Jun Huang. ETHICS DECLARATIONS COMPETING INTERESTS T.J.H. has co-founded a

start-up company, Ascent Bio-Nano Technologies Inc., to commercialize technologies involving acoustofluidics and acoustic tweezers. All other authors declare no competing interests. PEER

REVIEW PEER REVIEW INFORMATION _Nature Communications_ thanks Per Augustsson, and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file

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_et al._ Joint subarray acoustic tweezers enable controllable cell translation, rotation, and deformation. _Nat Commun_ 15, 9059 (2024). https://doi.org/10.1038/s41467-024-52686-8 Download

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