TECHNOLOGIES FOR PARTICLE MANIPULATION USING HARMONIC ACOUSTIC WAVES

Technologies for harmonic acoustic manipulation of colloidal particles include a system having a piezoelectric substrate coupled to one or more segmented acoustic transducers and a fluid positioned above the substrate. The segmented transducers have multiple segments, each with a resonant frequency equal to a harmonic frequency. The system further includes a controller that generates a harmonic signal including multiple harmonic components and applies the signal to the segmented acoustic transducers to generate an acoustic potential field in the fluid and manipulate the colloidal particles. The system may translate or rotate the particles, and may form the particles into a colloidal crystal monolayer. The system may selectively pair or otherwise group and separate individual particles. The system may pair and separate multiple groups of particles. The system may measure adhesion between particles. The system may pattern particles over a surface. The colloidal particles may be cells.

BACKGROUND

The ability to assemble colloidal particles with well-controlled shapes and material properties has been studied as an excellent model for exploring how matter organizes in materials science, condensed-matter physics and biophysics. Unlike nanoparticles, microscale particles cannot easily self-assemble into high-quality crystals. To direct the colloidal assembly of microscale particles, various methods including the use of specific surface functionalities, such as DNA linkers and attractive “patches,” different liquid solvents, complex anisotropic particles, or the modification of colloids using phototactic, electric and magnetic mechanisms, have been reported. Typical approaches may have synthetic difficulties associated with specific colloidal shapes or materials, poor control and tunability of interactions, and may be difficult to generalize. Additionally, these colloid manipulation approaches may not be directly applied to cell manipulation applications to understand cell-cell interactions or build ordered biological structures.

Acoustic tweezers, which are the acoustic analogue of optical tweezers, eliminate the need for optical tables, high-powered lasers, and complicated and time-consuming optical alignment, and offer a contact-free, highly biocompatible approach for performing particle manipulation. However, current standing wave-based acoustic tweezers and the recently developed acoustic force spectroscopy method can only trap and manipulate particles as a group, limiting their ability to control individual particles for precise colloidal assembly selectively. To overcome this limitation, phased array transducers and acoustic hologram methods have been developed to manipulate millimeter-scale particles individually.

SUMMARY

According to one aspect of the disclosure, a method for harmonic acoustic manipulation of particles comprises introducing particles into a fluid positioned over a first surface of a piezoelectric substrate; generating a first harmonic signal comprising a plurality of harmonic components; and applying the first harmonic signal to a first harmonic acoustic transducer, wherein the first harmonic acoustic transducer is coupled to the first surface of the piezoelectric substrate and spaced apart from the fluid. In an embodiment, the particles are cells, colloids, extracellular vesicles, or particles with a corresponding diameter between 1 nm and 1 cm.

In an embodiment, the method further comprises varying a parameter of one or more harmonic components of the plurality of harmonic components after introducing the particles into the fluid. In an embodiment, the parameter comprises a frequency, an amplitude, or a phase. In an embodiment, the method further comprises manipulating a single particle or a group of particles within the fluid by varying the parameter of the one or more harmonic components.

In an embodiment, manipulating the particles comprises translating a particle or a group of particles. In an embodiment, manipulating the particles further comprises rotating a particle or a group of particles. In an embodiment, manipulating the particles further comprises causing a plurality of particles to combine by reducing a distance between a corresponding acoustic trapping wells generated by the first harmonic signal. In an embodiment, manipulating the particles further comprises causing the plurality of particles to separate by increasing the distance between the corresponding pair of acoustic trapping wells generated by the first harmonic signal. In an embodiment, manipulating the particles further comprises measuring adhesion strength of a plurality of particles. In an embodiment, manipulating the particles further comprises c selectively manipulating target particles while keeping other particles intact. In an embodiment, manipulating the particles further comprises reducing a distance between a plurality of acoustic trapping wells generated by the first harmonic signal.

In an embodiment, applying the first harmonic signal comprises applying the signal with time-division multiplexing. In an embodiment, time-division multiplexing comprises, for each time division of the first harmonic signal, applying a signal with an excitation frequency corresponding to a resonant frequency of a segment of the first harmonic acoustic transducer.

In an embodiment, the piezoelectric substrate comprises lithium niobate, lithium tantalite, gallium arsenide, aluminum nitride, zinc oxide, lead zirconate titanate, or quartz. In an embodiment, the first harmonic signal has a fundamental frequency between 20 KHz and 10 GHz.

According to another aspect, a system for harmonic acoustic manipulation comprises a piezoelectric substrate having a first surface, and a segmented acoustic transducer coupled to the first surface of the piezoelectric substrate. The segmented acoustic transducer comprises a plurality of segments including a first segment and a second segment. The first segment has a first resonant frequency and the second segment has a second resonant frequency, wherein the second resonant frequency is an integer multiple of the first resonant frequency.

In an embodiment, each segment of the plurality of segments comprises a plurality of interdigital fingers separated by a finger pitch distance, wherein the first segment has a first finger pitch distance and the second segment has a second finger pitch distance, wherein the first finger pitch distance is the integer multiple of the second finger pitch distance. In an embodiment, the first finger pitch distance is twice the second finger pitch distance; and the second resonant frequency is twice the first resonant frequency.

In an embodiment, the system further comprises a first pair of segmented acoustic transducers coupled to the first surface of the piezoelectric substrate, the first pair including the segmented acoustic transducer; and a second pair of segmented acoustic transducers orthogonal coupled to the first surface of the piezoelectric substrate and orthogonal to the first pair.

In an embodiment, the system further comprises a controller configured to generate a first harmonic signal comprising a plurality of harmonic components, wherein each of the harmonic components has a frequency that corresponds to the resonant frequency of a segment of the segmented acoustic transducer; and apply the first harmonic signal to the segmented acoustic transducer.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring now toFIG.1, an illustrative system100for harmonic acoustic particle manipulation includes a piezoelectric substrate102coupled to multiple segmented harmonic interdigital transducers104. One potential embodiment of such a segmented transducer104is shown inFIG.3and further described below. A fluid layer106is positioned over the substrate102and between the transducers104. In use, and as described further below, a fluid including micro-scale particles such as cells or colloids is introduced to the fluid layer106, and one or more harmonic signals are applied to the transducers104. As described further below, those harmonic signals include multiple harmonic components, and the frequency of each harmonic corresponds to a resonant frequency of a particular segment of the segmented transducers104. The transducers104generate surface acoustic waves in the piezoelectric substrate102, which propagate underneath the fluid layer106. The surface acoustic waves couple with the fluid layer106and generate an acoustic potential field within the fluid layer106. The acoustic potential exerts gentle actuation forces on the particles suspended in the fluid layer106. Varying parameters of the harmonic signals allows for dexterous manipulation of the particles, without causing damage to fragile or soft particles such as cells or colloids. For example, the system100is capable of performing manipulations such as generating colloidal crystal monolayers or soft condensed matter, selectively pairing and separating single colloids, high-throughput pairing and separation of many pairs of colloids, high-throughput measurement of adhesion between colloids, patterning surfaces with colloids, and other manipulations.

One aspect of the present disclosure provides systems and methods using harmonic acoustics for non-contact, dynamic, and selective particle manipulation. Time-effective Fourier-synthesized harmonics are used to achieve such manipulation for the generation of reconfigurable acoustic lattices and spatial control of particles and cells suspended in liquid. The system100described herein can produce formation, reconfiguration, and precise rotational control of colloidal crystals or soft condensed matter. It can also actively control the lattice constant by the frequency or amplitude modulation of multi-harmonic waves to pair two target cells selectively with tunable inter-cellular distances or collectively manipulate an array of colloidal clusters or cells. It can control cells with more than 100 pairs in a suspension for reversible pairing and separation in a high-throughput, precise, programmable, and repeatable (>1,000 cycles) manner, all of which are improvements over existing colloid assembly or single-cell manipulation methods. With its soft yet powerful, precise yet high-throughput particle manipulation mechanism, the system100described herein provides a practical solution to provide deeper insights into intercellular adhesion forces, predict cancer metastasis, and establish a platform for personalized medicine via precision 3D biomaterial synthesis for organoid engineering.

Additionally, in comparison to existing techniques, the system100provides a more versatile method that can precisely manipulate both colloid materials and cells, without any surface treatment or modification of the particles' material properties, into desired formations. Further, the system100can be used to select single cells (roughly 10 μm diameter) or micrometer-scale colloidal particles, which may not be possible for typical systems due to their millimeter-level spatial resolution. Additionally, the system100provides precise assembly of colloidal matter and reversible cell-cell pairing and separation as compared to typical systems, which require either adjusting the phase or moving the transducer, and may have difficulty with precise assembly of colloidal matter and with reversible cell—cell pairing and separation due to the steady-state nature of the acoustic wavefield and/or the imprecision of acoustic streaming or vortex generation.

In the illustrated embodiment shown inFIG.1, the system100includes the piezoelectric substrate102, which is illustratively formed from a single-crystal piezoelectric material. Illustratively, the substrate102is formed from 128° Y-cut lithium niobite (LiNbO3). In other embodiment, the substrate102may be formed from any other appropriate piezoelectric material, including but not limited to lithium tantalite, gallium arsenide, aluminium nitride, zinc oxide, lead zirconate titanate, quartz, or other materials with piezoelectric properties. The fluid layer106is formed on the piezoelectric substrate102. The fluid layer106may be embodied as a liquid droplet, a fluid contained in a confined channel such as a microchamber, or other fluid layer positioned on the piezoelectric substrate102. Illustratively, the fluid layer106is contained in a microchamber formed from polydimethylsiloxane (PDMS) using soft lithography techniques. The illustrative microchamber has dimensions of 400×400×20 μm3, however in other embodiments the fluid layer106may have other micro-scale dimensions.

As shown, the system100includes four segmented interdigital transducers (IDTs)104athrough104d. In particular, the system100includes a pair of opposed segmented IDTs104a,104band another pair of opposed segmented IDTs104c,104dthat are positioned orthogonal to the segmented IDTs104,104b. The illustrative segmented IDTs104may be formed through photolithographic deposition of interdigital electrodes on the substrate102. As described further below, application of a harmonic electrical signal to each segmented IDT104generates surface acoustic waves in the piezoelectric substrate102through the piezoelectric effect. Although illustrated as including four IDTs104athrough104d, it should be understood that in other embodiments the system100may include a different number of segmented IDTs104, such as a single segmented IDT104or two segmented IDTs104. Each of the pairs of segmented IDTs104a,104band104c,104dmay operate as an acoustic transducer set, which is a collection of one or more acoustic transducers for generating acousto waves. Additionally, although the pairs of segmented IDTs104a,104band104c,104dare illustrated as being orthogonal to each other, it should be understood that in other embodiments the segmented IDTs104may be positioned at a different angle relative to each other (e.g., between 0 and 179 degrees). Additionally or alternatively, although illustrated as segmented IDTs104, it should be understood that in other embodiments, the system100may include any other acoustic transducer104capable of generating harmonic acoustic waves in the piezoelectric substrate102.

Referring now toFIG.2, one illustrative embodiment of a segmented IDT104is shown. The illustrative segmented IDT104includes a pair of electrodes202,204, which in the illustrative embodiment are formed from a 10 nm chromium (Cr) adhesion layer and an 80 nm gold (Au) conductive layer deposited onto the substrate102. As shown, each electrode202,204includes a respective rail206,208extending roughly parallel. Each electrode202,204further includes respective interdigitated fingers210,212extending toward the other electrode. More particularly, electrode202includes multiple fingers210extending roughly perpendicular from the rail206toward the rail208of the electrode204. Similarly, electrode204includes multiple fingers212extending roughly perpendicularly from the rail208toward the rail206of the electrode202. As shown, each finger210,212is separated from the next corresponding finger210,212by a pitch width214(shown only with respect to the electrode202to enhance clarity). As shown, except for fingers210,212on the end of the IDT104, each finger210,212from a particular electrode202,204is surrounded by fingers210,212from the other electrode202,204. That is, except for the ends, each finger210is surrounded by a pair of fingers212, and each finger212is surrounded by a pair of fingers210.

As shown, the segmented IDT104further includes multiple segments216. Illustratively, the IDT104includes three segments216a,216b,216c. For each segment216, the corresponding fingers210,212are separated by a different pitch width214. For example, in the segment216a, the fingers210a,212aare separated by the pitch width214a, in the segment216b, the fingers210b,212bare separated by the pitch width214b, and in the segment216c, the fingers210c,212care separated by the pitch width214c. The pitch width214of each segment216may be determined as a base width (e.g., the widest pitch width214a) divided by an integer associated with the segment (e.g., n=1, 2, . . . to N). For example, in the illustrative embodiment, the pitch width214bis one-half of the pitch width214a, and the pitch width214cis one-third of the pitch width214a. Conversely, each pitch width214may be determined as an integer multiple of the smallest pitch width214(e.g., the pitch width214bis twice the pitch with214c, and the pitch width214ais three times the pitch width214c). The base pitch width214amay be determined based on the wavelength of SAWs in the substrate102, for example as one-quarter wavelength of a standing wave in the substrate102. Although illustrated as including three segments216a,216b,216c, it should be understood that in some embodiments, the segmented IDT104may include a different number of segments216, such as six segments216, eight segments216, or another number of segments216. Further, although the illustrative segmented IDT104includes the same number of fingers210,212in each segment216, in some embodiments the segments216may have varying numbers of fingers210,212.

Accordingly, the segmented transducer104as shown inFIG.2is capable of generating multi-harmonic surface acoustic waves (SAWs). In particular, each individual segment216provides a distinct passband with wavenumbers [kn]Nat the resonant frequencies [fn]N, where N is the total number of segments216. Further, the intensity response of each harmonic component can be individually controlled with the modulation of electrode finger-pair 210, 212 numbers. Specifically, the response intensity can be strengthened for particular components by simply increasing the corresponding finger-pair 210, 212 numbers.

Referring again toFIG.1, each of the IDTs104is coupled to a signal source108, which may be embodied as a function generator, an oscillator, an amplifier, a variable-frequency signal generator, a digital-to-analog (D/A) converter, or any other signal source capable of generating a varying electrical signal. Illustratively, a signal source108ais coupled to the pair of IDTs104a,104b, and another signal source108bis coupled to the pair of IDTs104c,104d. In the illustrative embodiment, the electrodes of each pair of IDTs104a,104band104c,104dare coupled to the respective signal sources108a,108bsuch that electrodes of the same polarity relative to the signal source108are positioned opposite each other (i.e., connected in a cis-configuration). In some embodiments, the electrodes of each pair of IDTs104a,104band104c,104dare coupled to the respective signal sources108a,108bin a trans-configuration; that is, the polarity of the respective signal source108a,108bmay be reversed between the pairs of IDTs104a,104band104c,104d.

The signal sources108are coupled to a controller110, which may be embodied as a microcontroller, a digital signal processor, a programmable logic unit, a computer, or any other control circuit capable of controlling operations of the signal sources108. For example, the controller110may be capable of controlling one or more parameters of each signal source108, such as amplitude (i.e., voltage), frequency, phase, on/off time, or other parameters. To do so, the controller110may include a number of electronic components commonly associated with units utilized in the control of electronic and electromechanical systems. For example, the controller110may include, amongst other components customarily included in such devices, a processor and a memory device. The processor may be any type of device capable of executing software or firmware, such as a microcontroller, microprocessor, digital signal processor, or the like. The memory device may be embodied as one or more non-transitory, machine-readable media. The memory device is provided to store, amongst other things, instructions in the form of, for example, a software routine (or routines) which, when executed by the processor, allows the controller110to dynamically control parameters of the signal sources108using the other components of the system100. In some embodiments, the controller110may also include an analog interface circuit, which may be embodied as any electrical circuit(s), component, or collection of components capable of performing the functions described herein. The analog interface circuit may, for example, convert signals from the processor into output signals which are suitable for controlling the signal sources108. It is contemplated that, in some embodiments, the signal sources108(or portions thereof) may be integrated into the controller110.

In use, the electrical signals applied by the sources108to the IDTs104generate acoustic waves112that propagate through the piezoelectric substrate102. Illustratively, the opposing IDTs104a,104bgenerate opposing acoustic waves112a,112b, and similarly, the opposing IDTs104c,104dgenerate opposing acoustic waves112c,112d. The acoustic waves112a,112bare orthogonal to the acoustic waves112c,112d. As described further below, the acoustic waves112superimpose and otherwise interact to generate an acoustic potential field114within the fluid layer106.

In some embodiments, manipulation of particles within the fluid layer106may be monitored using an inverted microscope. The microscope image may be focused onto a camera (e.g., a CMOS sensor device) or other imaging sensor for video and/or still image recording. In some embodiments, a polarize chip may be used to eliminate double images caused by reflections from the substrate102surface.

Referring now toFIG.3, in use, the system100may execute a method300for dynamically manipulating particles with harmonic acoustic waves. It should be appreciated that, in some embodiments, one or more operations of the method300may be performed by the controller110and/or by other components of the system100as shown inFIG.1. The method300beings with block302, in which particles are introduced to the fluid layer106of the system100. The particles may be embodied as micro-scale particles or colloids suspended in a fluid medium. For example, the particles may include cells such as cancer cells, human cells, mouse cells, extracellular vesicles, or other biological colloidal particles. The particles may have a micro-scale size, for example an average diameter of about 10 μm, or about 2 μm, or between 5 μm and 15 μm, or between 1 μm and 20 μm, or between 1 μm and 1,000 μm, or between 1 nm and 1 cm.

In block304, the controller110generates multiple harmonic components for one or more surface acoustic wave (SAW) signals. The controller110may, for example, generate a Fourier-synthesized harmonic signal based on a predetermined number of harmonic frequencies. The controller110may generate six, eight, or a different number of harmonic components. As described above, each of those harmonic components may have a harmonic frequency that corresponds to the resonant frequency or otherwise matches a passband of a segment216of the segmented IDT104. The controller110may generate a single harmonic signal, for example for one-dimensional manipulation by a single IDT104or a single set of opposed IDTs104, two harmonic signals for two corresponding orthogonal sets of opposed IDTs104, or a different number of harmonic signals. When applied to the segmented IDT(s)104, the harmonic signals generate an acoustic potential field within the fluid layer106.

In block306, the controller110manipulates the acoustic potential field by varying one or more parameters of one or more harmonic components of the harmonic signals. The acoustic potential field may define one or more acoustic wells, which are regions where the acoustic radiation force is at a minimum. These acoustic wells may function as a node for trapping objects. Additionally or alternatively, in some embodiments certain objects may be trapped at antinodes or other regions where acoustic radiation force is at a maximum. The controller110may vary parameters of the harmonic components in order to change the location, size, shape, or other properties of one or more acoustic wells defined in the acoustic potential field. In some embodiments, in block308the controller110may modulate the frequency of one or more harmonic components, including the fundamental frequency and subsequent harmonic frequencies. For example, in some embodiments, the fundamental frequency may be changed from about 40 MHz to about 38 MHz for one set of orthogonal transducers104, which may change the shape of the acoustic wells. In some embodiments, in block310the controller110may modulate the amplitude of one or more harmonic components. For example, in some embodiments, the harmonic signal may have a fundamental frequency of 40 MHz and a second harmonic of 80 Mhz. The controller110may decrease the amplitude of the fundamental frequency and increase the amplitude of the second harmonic, which may reduce spacing between the acoustic wells. In some embodiments, in block312the controller110may modulate a phase difference of one or more harmonic components.

In block314, the controller110applies the harmonic signals to the segmented transducers104with time-division multiplexing to generate SAWs in the substrate102and thereby the fluid layer106. To perform the time-division multiplexing, the controller110may generate a signal at the particular harmonic frequency associated with each harmonic component for part of a small time division. Those time divisions may be small, for example on the order of nanoseconds. Thus, for micro-scale colloidal particles such as cells, the resultant acoustic waves are time-effective Fourier-synthesized harmonic SAWs. The controller110may, for example, cause a voltage source108to apply an electrical signal corresponding to the time-division multiplexed signal to the transducers104. In some embodiments, in block316, for each time division, the controller110selects an excitation frequency from the resonant frequencies of the segmented IDT104. In some embodiments, in block318the controller110may modulate the pulse duration for each harmonic component. In some embodiments, in block320the controller110may modulate the time division and ratio of active times for each frequency component. After applying the harmonic signals to the transducers104, the method300loops back to block306, in which the controller110may continue to manipulate the acoustic potential field by varying parameters of the harmonic components.

Referring now toFIG.4, diagram400shows illustrative experimental results for one-dimensional manipulation of particles that may be performed using the system100. Image402shows particles that are trapped in acoustic wells defined in the acoustoelectric field114. Illustratively, the particles are U937 cells. The image402is generated by fluorescent imaging and shows the fluorescent intensity profile averaged from five cell groups. Curve404illustrates the fluorescent intensity profile for the captured cells. As shown, a pair of cells406,408are captured in acoustic wells separated by about 30 μm. As described above, parameters of the harmonic signals applied to the IDTs104may be varied in order to selectively change the position of acoustic wells in the acoustoelectric field114. Image402′ shows the particles that are trapped in the acoustic wells defined in the modified acoustoelectric field114′. Curve404′ shows the modified fluorescent intensity profile averaged from the five cell groups. As shown, as the acoustic wells are moved closer together, the cells406,408have also been moved closer together, assembling a pair. Other cells in the other acoustic wells have not been moved. Accordingly, the system100is capable of selectively pairing cells and other colloidal particles at micrometer-scale resolution. Similarly, the system100is capable of selectively separating pairs of cells and other colloidal particles by modifying the acoustic potential field114in reverse. The system100is further capable of reversibly pairing and separating pairs of cells and other colloidal particles.

Referring now toFIG.5, diagram500shows illustrative experimental results for two-dimensional manipulation of particles that may be performed using the system100. Image502shows particles504that are trapped in acoustic wells defined in the acoustic potential field114. Illustratively, the particles504are 2 μm fluorescent polystyrene particles. As shown in the image502, the particles504are trapped in acoustic wells with equal trapping spacing, with each well separated by a distance506, which is illustratively about 30 μm. The spacing between trapping wells can be tuned by modifying parameters of the harmonic components as described above. Image502′ shows the particles504trapped in a modified acoustic potential field114′. As shown, there is a tuneable distance between trapping wells, with certain trapping wells separated by distance508and other wells separated by a trapping distance510. Illustratively, the distance510is 1.5 times the distance508(e.g., about 45 μm and about 30 μm, respectively). The distances between trapping wells is tuneable and may be varied over time by varying the harmonic components of the harmonic signals used to drive the IDTs104.

Referring now toFIGS.6and7, diagrams600,700shows illustrative experimental results for colloidal crystal monolayer manipulation of particles that may be performed using the system100. As shown in subdiagram602of the diagram600, initially a group of particles604are randomly distributed throughout the fluid layer104. The particles604may be cells or other colloidal particles. As shown in subdiagram606, after application of an acoustic field potential114, the particles604form clumps at locations of trapping wells. The illustrative acoustic field potential114may be formed by standing SAWs in the x- and y-directions on the substrate102. Subdiagram608shows the particles604after applying a modified acoustic field potential114′ generated using harmonic SAWs as described above. As shown, the acoustic field potential114′ generates focusing forces610in the z-direction, which moves the particles604into a crystalline monolayer. As shown in the diagram700ofFIG.7, when the modified acoustic field potential114′ is applied, the particles604are arranged in a compact crystalline structure. This crystalline structure spins in the x-y plane about an axis that lies in the z-direction, as shown by the arrow of rotation702. The direction and speed of the rotation may be controlled by modifying the phase difference of the harmonic signals. Additionally, particles604may move into compact crystalline positions as the colloidal crystal structure rotates. For example, particle604amay move to a final position, illustrated in phantom as particle604a′. Spin speed may also depend on other factors, such as the configuration of particles604in each colloidal crystal monolayer and/or the number of particles604in each colloidal crystal monolayer.

Referring now toFIG.8, diagram800shows additional illustrative experimental results for colloidal crystal monolayer manipulation of particles that may be performed using the system100. The diagram800shows imaging results for experiments performed using fluorescent polystyrene particles having a diameter of 2 μm. As shown, crystals with multiple different particle numbers and shapes may be formed. For example, image802shows a crystal with particle number equal to three, image804shows a crystal with particle number equal to four, image806shows a crystal with particle number equal to six, image808shows a crystal with particle number equal to seven, image810shows a crystal with particle number equal to eight, image812shows a crystal with particle number equal to nine, image814shows a crystal with particle number equal to 10, image816shows a crystal with particle number equal to 12, image818shows a crystal with particle number equal to 14, image820shows a crystal with particle number equal to 18, image822shows a crystal with particle number equal to 19, and image824shows a crystal with particle number equal to 24.

By tuning the frequencies and amplitudes of five-component harmonic SAWs (such as f2to f6), the shapes and sizes of the harmonic acoustic wells can be modulated, which enables the generation of diverse crystal monolayers with different numbers of particles. Due to the secondary acoustic radiation forces generated by the scattering of the acoustic field between the particles, the monolayer assemblies can be stabilized as close-packed colloidal crystals. Manipulation as described herein can also spin the entire colloidal crystal assembly by applying a phase difference (Δφ=φ×−φy) between the x and y direction harmonic SAWs. This acoustic-induced rotation allows creation of various colloidal crystals from the initial cluster by further shaping the colloidal assemblies with the stabilized monolayer patterns. Additionally, the rotational direction of an assembly can be easily tuned by changing the phase difference of the applied harmonic SAWs. For example, a positive phase difference (Δφ>0) may result in a clockwise rotation and a negative phase difference (Δφ<0) may result in a counterclockwise rotation for a ten-particle cluster monolayer. After the further quantitative study, it has been demonstrated that the rotational speed of the colloidal assembly is linear with respect to the phase difference, and this speed can be proportionally tuned by varying the amplitudes of the applied excitations.

Additionally, spin speed of the colloidal monolayers with different numbers of particles (from n=2 to n=10) and configurations (four monolayer configurations with n=6) was investigated. Under the same excitation, it was observed that the spin speed of the assemblies is strongly correlated with the colloidal crystal configurations rather than with the particle numbers. For example, for certain investigated configurations of six-particle clusters, the highest spin speed is achieved by a flower-shaped arrangement with the rotational symmetry of order five. This observation suggests that clusters with higher orders of rotational symmetry tend to have a faster spin speed. It also demonstrates the capability of manipulation by the system100for different crystal configurations of the colloidal crystal monolayer by varying the spin speeds. With this capability for precise particle assembly, the system100may be used to explore the fundamental soft condensed-matter physics behind colloidal interactions and assembly.

Referring now toFIG.9, diagram900shows illustrative experimental results that may be achieved for high-throughput particle combination and separation. Image902shows multiple colloidal particles trapped in an acoustic potential field114. The illustrative particles may be cells or other colloidal particles having a diameter of 9.51 μm. Each particle may be trapped in an acoustic well, and the wells may be isolated from each other or connected in a mesh-like arrangement. As described above, the acoustic potential field can be actively controlled in order to manipulate the particles. For example, by switching from second harmonic (80 MHz) to base (40 MHz) frequencies, the acoustic potential field pattern114may be reconfigured from single-colloid trapping to pair trapping. As another example, by using harmonics (39.8 MHz) in one direction that are slightly shifted from harmonics (40 MHz) in the orthogonal direction, the acoustic field pattern114can form a uniform rectangular pattern that enables repeatable switching between trapping of a single colloid and pairing colloids. The diagram900illustrates such pairing. For example, as the acoustic potential field changes, particles904may experience forces906causing the particles904to form a pair. Similarly, particles908may also experience forces910causing the particles908to form a pair. Image902′ shows the particles904,908after pairs have been formed. As shown, the particles may be paired with particles from different directly. Those pairs may be separated by reversing the direction of the change in acoustic potential field. Accordingly, the system100may reversibly pair and/or separate colloids with high throughput (e.g., more than 100 simultaneous pairs).

Thus, the distance between individual colloids or cells can be dynamically modulated with subwavelength manipulation resolution. This subwavelength manipulation resolution is achieved with a time-effective Fourier-synthesized acoustic potential field that was realized by sequentially applying nanosecond pulsing of SAWs with a time-division multiplexing method during a period T By modulating the time-division Δt1, Δt2and their ratio κ, spectrum trapping occurs and enables positional tuning to precisely manipulate objects in a half-wavelength range of applied SAWs. On the basis of analytical simulations, this spectrum trapping method can provide spatial control with nanometer precision. By shaping the acoustic potential field on demand, the configuration of an acoustic-well array can be actively controlled. For example, a mesh-like arrangement of connected acoustic wells can be generated by using the same frequency to excite standing SAWs in the x and y directions. By switching from the second harmonic (fx2=fy2=80.0 MHz) to base (fx1=fy1=40.0 MHz) frequencies, the lattice constant of the acoustic-well array changed from (√{square root over (2)}/4)λ1to (√{square root over (2)}/2)λ1, causing the reconfiguration of the pattern from single-colloid trapping to pair. Specifically, at higher harmonics, each colloid occupies an acoustic well. However, the number of acoustic wells is reduced with a decrease in frequency, which forces the colloids to settle the same acoustic well at lower harmonics. When using harmonics (fx1=39.8 MHz) in the x direction that are slightly shifted from the harmonics (fy1=40.0 MHz) in the y direction, a dot-like array of isolated acoustic wells can form a uniform rectangular pattern that enables repeatable switching between trapping of a single colloid and pairing. Notably, via dynamic switching among nanosecond pulsing harmonics, a time-effective Fourier-synthesized acoustic potential field can be formed. Furthermore, the generated harmonic acoustic wells can be programmed with tunable sizes and spacings between neighboring wells. To demonstrate the dynamic patterning capability of the system100, customized colloid patterns were created that form the shapes of the letters, ‘O,’ ‘D,’ and ‘K’, respectively, via modulating the five-component harmonic SAWs, as described further below in connection withFIG.11.

By dynamically and reversibly switching between the single-colloid trapping and pairing modes, repeatable (operating for more than 1,000 pairing cycles) and high-throughput (>100 pairs simultaneously) studies can be performed. The separation force during this reversible pairing process was characterized for polystyrene particles with an average diameter of 9.51 μm. The force—time curves were experimentally measured, and the peak separation force was calculated to range from 1.6 to 19.5 pN with variable excitation amplitudes. Note that the separation force scales with the square of the SAW excitation amplitudes. The separation force applied on cells can also be calculated. The time for particles to be fully separated was approximately 12 ms when using an 8 V SAW excitation signal. Combining this short exposure time with the piconewton separation forces decreases the likelihood that this acoustic manipulation method would interfere with cell sample measurements.

Additionally, selective manipulation for single cell—cell pairings was investigated. Individual U937 cells (marked with different colours) were trapped in four adjacent acoustic wells. By switching the frequency of the harmonic standing SAWs between 39.8 and 79.6 MHz in the x direction, while keeping the frequency constant at 40.0 MHz in the y direction, two pairs of U937 cells can be periodically brought into contact and then separated in the x direction. To perform reversible cell—cell pairing in the y direction, swapped the applied excitations may be swapped in the x and y directions. It was demonstrated that each cell could be paired with cells from different directions. Furthermore, U937 cells can be selectively paired while keeping neighboring cells intact by modulating the synthesized six-component harmonic SAWs. In summary, the system100can reversibly pair cells in a high-throughput manner and can also selectively target any two neighboring cells by modifying the applied multi-harmonic waves.

Referring now toFIG.10, diagram1000shows illustrative experimental results that may be achieved for measuring adhesion strength for combined particles. In the experiment, cells were repeatedly paired and separated using similar techniques as described above. Chart1002illustrates results for an experiment using M0 THP-1 cells. Curve1004represents the acoustic potential field114. When the curve1104is at the low value, the acoustic potential field114forces the cells to pair, and when the curve1104is at the high value, the acoustic potential field114forces the cells to separate. Curve1006illustrates normalized distance between pairs of cells. As shown, the curve1006can be separated into three phases1008,1010,1012. In the phase1008, the paired cells approach each other, in the phase1010the cells contact each other, and in the phase1012the cells retract from each other.

Chart1014illustrates results for another experiment using M1 THP-1 cells. Curve1004represents the acoustic potential field114, similar to the chart1002. Curve1016illustrates normalized distance between pairs of cells. As shown, the curve1016can be separated into four phases1008,1010,1018,1012. In the phase1008, the paired cells approach each other, in the phase1010the cells contact each other, in the phase1018the cells adhere to each other, and in the phase1012the cells retract from each other. As shown, the M0 THP-1 cells are non-adherent, and the M1 THP-1 cells are adherent. The length of the phase1018, or the adhesion lifetime, may depend on the balance between separate force caused by the acoustic potential field114and adhesion force between the cells. Accordingly, the system100is capable of measuring and distinguishing adhesion differences between adherent and non-adherent cells. Similarly, the system100may measure adhesion differences caused by drug treatments or other treatments of the cells. As another example, the system100may distinguish between groups of cells or cell lines. For example, the system100may use measured adhesion strength to distinguish and/or identify metastatic cancer cells as compared to other cells.

The ability to distinguish variations in cell-cell adhesion is a significant quantitative capability for any single-cell manipulation and analysis technique. Typical techniques, such as atomic force microscopy, micropipette aspiration and optical tweezers, may require direct physical contact with the cells. Additionally, these techniques can typically only probe one cell pair each time, which is time-consuming and labor-intensive for any practical assay. With the ability to simultaneously perform repeated and reversible cell-cell pairing in a large array in suspension as described above, cell-cell adhesion assays were conducted with the system100on various cell lines. As described above, it was investigated whether the system100could distinguish adhesion differences between adherent THP-1 macrophages (M1 THP-1) and non-adherent THP-1 monocytes (M0 THP-1). Here, THP-1 macrophages are differentiated from THP-1 monocytes with phorbol-12-myristate-13-acetate (PMA) as a stimulus. As shown inFIG.10, non-adherent M0 THP-1 cells begin their retraction period immediately after a 2 s contact signal is deactivated, since their intrinsic adhesion forces are not adequate to balance the acoustic separation forces. Conversely, M1 THP-1 cells cannot be immediately separated due to the significantly increased adherence with the PMA stimulation. The cell-cell distance trace for M1 THP-1 cells is a flat line for the period immediately after contact signal deactivation. This is due to the force clamp (the balance between the separation force and the adhesion force), and the duration of this force clamp period is defined as the adhesion lifetime (t1). Using this value, differences in adhesion forces between cell types can be distinguished consistently and quantitatively. Additionally, the applied contact signal duration tcswas varied (0.5-2.5 s) to investigate variations in adhesion lifetimes. For example, both heatmaps of the adhesion lifetimes and histograms of the adhesion frequency (defined here as the percentage of adhesion lifetime events per cell pair for 50 contact and separation cycles) present that the adhesion strength of M1 THP-1 cells increased with the duration of the applied contact signal. A western blot analysis with a high expression of the intercellular adhesion molecule ICAM-1 confirmed that there should be a higher cell-cell adhesion strength in the M1 THP-1 cells than that in the M0 THP-1 cells. Altogether, these results support that the system100can successfully measure and distinguish adhesion differences between adherent and non-adherent cells.

In addition to cell surface proteins, cell-cell adhesion strength could also be affected by other intrinsic properties of cells, such as the actin cytoskeleton organization. Thus, the system100was applied to examine and quantify the variation of cell-cell adhesion strength caused by perturbations in the organization of the actin cytoskeleton. Adhesion differences were explored in MCF-7 cells with and without a Cytochalasin D (CytoD) treatment. The CytoD treatment affects the organization of the cytoskeletal network and inhibits actin polymerization in cells. Previous experiments have demonstrated that actin polymerization regulates the rapid cell-cell adhesion during cell migration. Without the CytoD treatment (CytoD−), the histogram of the adhesion lifetimes for MCF-7 cells shows a bimodal distribution of short (tshort) and long (tlong) lifetimes with average values of 0.30±0.15 and 0.82±0.15 s, respectively, as determined by a Gaussian fit. In sharp contrast, CytoD+ MCF-7 cells had a lower fraction (34.2 versus 62.3%) of long lifetimes than CytoD− MCF-7 cells, which indicates a reduction in adhesion strength after CytoD treatment.

Since intercellular adhesion forces are critical information about cell—cell attachment and detachment, the capability of the system100to quantify the variations in cell adhesion forces among different cell lines (SupplementaryFIG.5i) was investigated. MDA-MB-231 and human embryonic kidney 293T (HEK293T) cells were tested following the same protocol as used with the MCF-7 cells. Compared with CytoD− MCF-7 cells, MDA-MB-231 cells show a lower fraction (41.5 versus 62.3%) of long adhesion lifetimes, which suggests a lower cell adhesion than CytoD− MCF-7 cells. Additionally, the adhesion lifetimes of HEK293T cells show an almost equally populated bimodal distribution with similar short (46.4%) and long (53.6%) lifetime subpopulations. For each cell line tested above, the bimodal Gaussian distribution in the lifetime enables a detailed comparison of the fractions of short and long lifetimes. The adherent cells may be compared on the basis of their fractions of lifetime subpopulations, and the average lifetime fraction ratio RL/S (the ratio of the long lifetime fraction to the short lifetime fraction) may be calculated for each cell type. This average lifetime fraction ratio could be used as an indicator for indirectly measuring the differences in cell—cell adhesion behavior and strength. Notably, the adhesion strength decreased (as indicated by RL/S decreasing from 1.65 to 0.71) as the metastatic potential increased for the human breast cancer cell lines from MCF-7 to MDA-MB-231, which is consistent with measurements taken by micropipette aspiration and static methods.

Referring now toFIG.11, diagram1100shows illustrative experimental results that may be achieved for patterning a surface with particles using the system100. Images1102a,1102b,1102cillustrate predicted acoustic potential fields114associated with particular harmonic signals. Illustratively, the harmonic signals are synthesized to create acoustic potential fields114having shapes corresponding to the letters “O”, “D,” and “K,” respectively. Images1104a,1104b,1104cillustrate particles1106that have been arranged on the substrate102by application of the acoustic potential fields114as described above. Illustratively, the particles1106are 2 μm polystyrene particles.

Referring now toFIG.12, diagram1200illustrates harmonic components and harmonic signals that may be generated by the system100. Chart1202illustrates one potential embodiment of a time-division multiplexed harmonic signal that may be generated by the controller110and applied to one or more segmented IDTs104. Curve1204represents amplitude of the electrical signal that maybe applied to the IDTs104. The curve1204includes a time division1206, which is a relatively short duration of nanosecond scale. The time division1206is further subdivided into harmonic components1208,1210,1212,1214,1216as described above. As shown, each harmonic component has as different harmonic frequency and has a particular amplitude as determined by the controller110. Each harmonic component may also have other varying parameters such as pulse width, phase, or other tonal parameters. The harmonic signal applied to the IDTs104includes repeated time divisions1206.

Chart1218illustrates example one-dimensional acoustic potential fields114that may be generated by the system100based on time-division multiplexed signals similar to those shown in the chart1202. In particular, curve1220represents an acoustic potential field that may be generated for the signal1204shown in the chart1202. The curve1220may correspond to an acoustic potential field for two-dimensional colloidal crystal monolayer generation as described above. Similarly, curve1222may correspond to an acoustic potential field for patterning particles as described above. Curve1224may correspond to an acoustic potential field for high-throughput particle pairing and separation as described above. Curve1226may correspond to an acoustic potential field for selective particle pairing and separation as described above.