Cell selective manipulation with single beam acoustical tweezers
Michael Baudoin, Jean-Louis Thomas, Roudy Al Sahely, Jean-Claude Gerbedoen, Zhixiong Gong, Aude Sivery, Olivier Matar, Nikolay Smagin, Peter Favreau, Alexis Vlandas
CCell selective manipulation with single beam acoustical tweezers.
Michael Baudoin,
1, 2, ∗ Jean-Louis Thomas, Roudy Al Sahely, Jean-Claude Gerbedoen, ZhixiongGong, Aude Sivery, Olivier Bou Matar, Nikolay Smagin, Peter Favreau, and Alexis Vlandas † Univ. Lille, CNRS, Centrale Lille, Yncra ISEN, Univ. Polytechnique Hauts-de-France,UMR 8520 - IEMN, SATT NORD, F- 59000 Lille, France. Institut Universitaire de France, 1 rue Descartes, 75005 Paris Sorbonne Universit´e, CNRS, Institut des NanoSciences de Paris, INSP, F-75005 Paris, France (Dated: April 20, 2020)Acoustical tweezers open major prospects in microbiology for cells and microorganisms contact-less manipulation, organization and mechanical properties testing since they are biocompatible,label-free and can exert forces several orders of magnitude larger than their optical counterpartat equivalent wave power . Yet, these tremendous perspectives have so far been hindered by theabsence of selectivity of existing acoustical tweezers - i.e., the ability to select and move objectsindividually - and/or their limited resolution restricting their use to large particle manipulationonly . Here, we report precise selective contactless manipulation and positioning of human cells ina standard microscopy environment, without altering their viability. Trapping forces of up to ∼ ) designed to produce stiff localized traps. We anticipate this work to be astarting point toward widespread applications of acoustical tweezers in fields as diverse as tissueengineering , cell mechano-transduction analysis , neural network study or mobile microor-ganisms imaging , for which precise manipulation and/or controlled application of stresses ismandatory. I. INTRODUCTION
Contactless tweezers based on optical and mag-netic forces have been developed in the last decadesand have led to tremendous progress in science recognizedby several Nobel prizes. Nevertheless, these technologieshave stringent limitations when operating on biologicalmatter. Optical tweezers rely on the optical radiationpressure, a force proportional to the intensity of the wave-field divided by the speed of light. The high value of thelatter severely limits the forces that can be applied andimposes the use of high intensity fields. This can leadto deleterious photothermal damages (due to absorptioninduced heating) and/or photochemical damages (due toexcitation of reactive compounds like singlet oxygen) adversely affecting cells integrity. Magnetic tweezers, onthe other hand, can only manipulate objects susceptibleto magnetic fields and thus require other particles to bepre-tagged with magnetic compounds, a limiting factorfor many applications. For biological applications, acous-tical tweezers are a prominent technology . Theyrely on the acoustical radiation force , which is -as fortheir optical counterpart- proportional to the intensityof the wave divided by the wave speed. But, the dra-matically lower speed of sound compared to light leadsto driving power several orders of magnitude smallerthan in optics to apply the same forces (or conversely,forces several orders of magnitude larger at the samedriving power) . In addition, the innocuity of ul-trasounds on cells and tissues below cavitation thresh-old is largely documented and demonstrated dailyby their widespread use in medical imaging . Indeed, the frequencies typically used in ultrasound applications(100 kHz to 100 MHz) are far below electronic or molecu-lar excitation resonances thus avoiding adverse effects oncells integrity. Moreover, the weak attenuation of soundin both water and tissues at these frequencies limits ab-sorption induced thermal heating. Finally, almost anytype of particles (solid particles, biological tissues, drops)can be trapped without pre-tagging and the low speedof sound enables spatial resolution down to micrometricscales even at these comparatively low frequencies.Nevertheless, the promising capabilities offered byacoustical tweezers have so far been hindered by the lackof selectivity of existing devices and/or their restrictedoperating frequency limiting their use to large particlesonly. Yet, the ability to select, move and organize in-dividual microscopic living organisms is of the utmostimportance in microbiology for fields at the forefront ofcurrent research such as single cell analysis, cell-cell in-teraction study, or to promote the emergence of disrup-tive research e.g. on spatially organized co-cultures. Inthis paper, we unleash the potential of acoustical tweez-ers by demonstrating individual biological cells manipu-lation and organization in a standard microscopy envi-ronment with miniaturized single beam acoustical tweez-ers. The strength and efficiency of acoustical tweezers isillustrated by exerting forces on cells one order of mag-nitude larger than the maximum forces reported withoptical tweezers ( ∼
200 pN), obtained with one orderof magnitude less wave power ( < a r X i v : . [ phy s i c s . b i o - ph ] A p r A Vortex T w ee z e r Cells Microfluidic chamber
CB D E Glass slide X , Y m o t o r i z e d s t a g e Inverted microscopeSpiraling tweezerRadial electrodes 2mm
Electricalconnections
21 mm
X YZ 47 MHz Glass substrate
FIG. 1. Experimental setup. A. Illustration of the working principle of the tweezers designed for cells selective manipulation:A spherically focused acoustical vortex is synthesized by spiraling active electrodes metallized at the surface of a piezoelectricsubstrate and actuated with a function generator connected to an amplifier. The vortex propagates and focalizes inside a gluedglass substrate and then reaches a microfluidic chamber made of a glass slide and a PDMS cover containing cells embedded ina growth medium. The microfluidic device is acoustically coupled with the transducer with a thin layer of silicone oil (25 cSt).A cell located at the center of the acoustical vortex is trapped. Its motion relative to other cells is enabled by the displacementof the microfluidic chamber driven by a XY motorized stage (See Movie 1 for an animated explanation of the setup workingprinciple). B. Picture of typical transducers used in the present study (right) and illustration of the scale reduction comparedto previous lower frequency designs by Baudoin et al . (left). C. Image of the actual experimental setup. D. Zoom in on thespiral transducer and the electrical connections (in black). E. Illustration of the integration of the whole setup inside a standardinverted microscope. Photo credit: B: J.-C. Gerbedoen, SATT NORD / C-D-E: R.A. Sahely, Univ. Lille. II. ACOUSTICAL TWEEZERS DESIGN
The first experimental evidence of large particles trap-ping with acoustic waves dates back to the early 20thcentury . Nevertheless, the first demonstration of con-trolled manipulation of micrometric particles and cellswith acoustic waves appeared only one century later withthe emergence of microfluidics and high frequency trans-ducers based on interdigitated electrodes . In these re-cent works, trapping relies on the 2D superposition oforthogonal plane standing waves, an efficient solution forthe collective motion of particles, but one which precludesany selectivity, i.e., the ability to select and move oneparticle out of a population . Indeed, the multiplicity ofnodes and anti-nodes leads to the existence of multipletrapping sites which cannot be moved independently.In addition, multiple transducers or reflectors positionedaround the manipulation area are mandatory for the syn-thesis of standing waves, a condition difficult to fulfill inmany experimental configurations.Selective trapping with single beam requires strongspatial localization and hence tight focusing of the wave-field. In optics, this ability has been achieved with fo-cused progressive waves , a solution also investigated in acoustics . But such wavefields are inadequate inacoustics for most particles of practical interest, sinceobjects with positive contrast factors (such as rigid par-ticles or cells) are attracted to pressure nodes andwould be expelled from the focal point of a focusedwave . Acoustical vortices provide an elegant solu-tion to this problem . These focused helical progres-sive waves spin around a central axis wherein the pres-sure amplitude vanishes, surrounded by a ring of highpressure intensity, which pushes particles toward thecentral node. Two-dimensional trapping and three-dimensional levitation and trapping have been pre-viously reported at the center of laterally and spheri-cally focused vortices, respectively. However, all thesedemonstrations were performed on relatively large par-ticles ( > µ m in diameter) using complex arrays oftransducers, which are cumbersome, not compatible withstandard microscopes, and that cannot be easily minia-turized to trap micrometric particles. Recently, Baudoin et al . demonstrated the selective manipulation of 150 µ m particles in a standard microscopy environment withflat, easily integrable, miniaturized tweezers. To reachthis goal, they sputtered holographic electrodes at thesurface of an active piezoelectric substrate, designed tosynthesize a spherically focused acoustical vortex.Nevertheless, transcending the limits of this technologyto achieve selective cells manipulation remained a majorscientific and technological challenge. Indeed, the systemshould be scaled down (frequency up-scaling) by a fac-tor of 10 (since cells have typical size of 10 µ m), whileincreasing dramatically the field intensity, owing to thelow acoustic contrast (density, compressibility) betweencells and the surrounding liquid . In addition, sincethe concomitant system’s miniaturization and power in-crease are known to adversely increase the sources ofdissipation, the tweezers had to be specifically designedto prevent detrimental temperature increase and enabledamage free manipulation of cells:First, spherically focused acoustical vortices (Fig. 1A)were chosen to trap the particles. Indeed, the energy con-centration resulting from the 3D focalization (Fig. 2F)enables to reach high amplitudes at the focus from re-mote low power transducers. These spherically focusedvortices were synthesized by materializing the hologramof a ∼
45 MHz vortex with metallic electrodes at the sur-face of an active piezoelectric substrat. The hologramwas discretized on two levels resulting in two intertwinedspiralling electrodes (Fig. 1D), patterned in a clean roomby standard photo-lithography techniques (see Methodssection A). The scale reduction compared to our previousgeneration of acoustical tweezers is illustrated in Fig.1B. Second, the design of the electrodes was optimizedto reduce Joule heating (magnified by the scale reduc-tion) inside the electrodes. To prevent this effect, (i) thethickness of the metallic electrodes was increased by afactor of 2 (400 nm of gold and 40 nm of titanium); (ii)the width of the electrical connections (Fig. 1D) supply-ing the power to the spirals was significantly increased toprevent any dissipation before the active region; and (iii)two radial electrodes spanning half of the spirals wereadded as a way to effectively bring power to the drivingelectrode. Third, a 1 . III. CHARACTERIZATION OF THEACOUSTICAL TRAP
The principle of high frequency acoustical vortices syn-thesis with these active holograms was assessed throughthe comparison of numerical predictions obtained froman angular spectrum code and experimental measure-ments of the acoustic field normal displacement at thesurface of the glass slide (XY plane) with a PolytechUHF-120 laser Doppler vibrometer (Fig. 2, A-D). Boththe intensity and phase are faithful to the simulations anddemonstrate the ability to generate high frequency acous-tic vortices. As expected, the wavefield exhibits a cen-tral node (corresponding to the phase central singularity)surrounded by a ring of high intensity which constitutesthe acoustical trap. The magnitude of the acoustic field(displacement) depends on the driving electrical powerand was measured to vary typically between 0 . µ W and 2 mW (see Methods section I). Theconcentration of the acoustic energy through focalizationin the propagation plane (XZ) can be seen in Fig. 2E.An estimation of the lateral force field exerted on a cellof 10 µ m radius with density 1100 kg m − and compress-ibility 4 × − Pa − was computed at each point inthe manipulation plane of the microfluidic chamber (XYplane, Fig. 2F) with the theoretical formula derived bySapozhnikov & Bailey . This calculation gives an esti-mation of the force of the order of 100 pN, which cannevertheless strongly vary depending on the exact cellsproperties (see Methods section D for the exact values de-pending on cells acoustic properties for an acousticvibration of 1 nm).Finally, the temperature increase due to Joule heat-ing in the electrodes as well as the total temperatureincrease due to both Joule heating and acoustic waveabsorption was measured using an infrared camera to as-sess potential impact on biological material (See Methodssection J). For most experiments presented in this pa-per (corresponding to acoustic displacement < . . ◦ C after 2 minof manipulation and even vanishes for the lowest power(0 . . ◦ C at thetop of the glass slide and 5 . ◦ C inside a drop of glyc-erol placed on top of the glass slide (acting as a perfectlyabsorbing medium) at the highest power used for highspeed displacement of the cells. These measurements in-dicate that the first source of heat is Joule heating inthe electrodes which could be solved by active coolingof the transducer. They also suggest that even at thelargest power used in the present experiments, the mod-erate temperature increase remains compatible with cellsmanipulation, as assessed in the next section.
A Intensity XY: numericalB Phase XY: numerical C Intensity XY: experimental -100 -50 0 50 100 x position ( m) -100-50050100 y po s i t i on ( m ) -3-2-10123 - - x po s i t i on ( m ) - -
50 0 50 100 y position ( m) - - - D Phase XY: experimental E Amplitude XZ: numericalF Lateral force field: numerical x position ( m) -1-0.500.51 N o r m a li z ed l a t e r a l f o r c e FIG. 2. Acoustic field and radiation forces. A-D. Numerical predictions (A and B) and experimental measurements (C and D)with a UHF-120 Polytec laser Doppler vibrometer of the normalized modulus (A and C) and phase (C and D) of the acousticnormal displacement at the surface of the glass slide (XY plane). In the experiments presented in this paper, the maximumamplitude of the vibrations (displacement) on the ring lies typically between 0 . . IV. CELLS MANIPULATION, POSITIONINGAND VIABILITY
Cell manipulation is demonstrated in a microfluidic de-vice integrated in a standard inverted microscope (Fig.1E) to illustrate the fact that our approach can be easilytransposed to standard microbiology experiments. Thedevice is composed of a thin glass slide treated to preventcell adhesion and a PDMS chamber of controlled height(38 µ m). The cells are loaded by placing a drop of thecell suspension (10-20 µ L) on the glass surface using amicro-pipette and carefully lowering the chamber on topof the drop. The position of the vortex core is spottedwith four triangular marks deposited at the surface ofthe glass substrate. Using a XY positioning system it isthereafter possible to align the tweezers center to any cellpresent in the chamber. Upon activation of the AC driv-ing signal, a cell situated inside the vortex core is nearlyinstantaneously trapped.The first demonstration of the selective nature of ourtweezers is showcased by our ability to pick up a singlecell (breast cancer cell MDA-MB-231, 7 ± µm in radius) amongst a collection of cells and move it along a slalomcourse where other free to move cells act as poles (see Fig.3A and Movie 2). Then a second cell initially serving as aslalom marker, is moved to prove that it was free (Movie2). The precise displacement can be performed in anydirection as demonstrated by the square motion of a cellaround another (Fig. 3B, Movie 3). Displacement can beperformed even in the presence of other cells without anyrisk of ”coalescence” as the first ring acts as a barrier. Ascan be seen in Fig. 2C, the radius of the first repulsivering is typically 40 µ m. The second ring of much weakerintensity can also slightly affect free cells at large power.One of the key ability enabled by acoustical tweezers isthe capture, positioning and release of cells at precise lo-cations. As an illustration, a total of 10 individual MDAcells were therefore positioned to spell the letter ”A” and”T” of ”Acoustical Tweezers” (Fig. 3C). The total ma-nipulation time to achieve these results was kept under10 min (less than 2 min per cell). All the operations rep-resented in Fig. 3 were performed with acoustic vibrationdisplacements < . B BCA 1 2 3 4 5 𝜇 m 50 𝜇 m50 𝜇 m FIG. 3. A. Stack of images illustrating the selective manipulation of a human breast cancer cell (MDA-MB-231) of radius 7 ± µ m between other cells. The blue dotted line and green continuous line show respectively the future and past path followed bythe cell. (See also Movie 2) B. Image illustrating the square relative motion of a trapped cell ”1” of 7 ± µ m (located in thecenter of the picture) around another cell ”2” obtained by superimposing the images of the two cells in the frame of reference ofthe trapped cell (see also Movie 3). In this frame of reference, the successive positions of cell ”2” form a square. For the sake ofclarity other cells appearing in the field of view have been removed. C. Manipulation of 10 MDA cells (average radius 9 µ m toform the letters ”A” and ”T” of ”Acoustical Tweezers”. Note that in these pictures the focus is voluntarily left under-focusedto improve contrast of the cells. the forces that can be exerted on cells with these tweez-ers. For this purpose, cells were trapped and thenmoved with an increasing speed until it was ejected fromthe trap. Velocities up to 1.2 mm s − before ejectionhave been measured for cells displacement of diameter12 ± µ m trapped with an acoustic field of magnitude0 . µ m (see Movie4). This corresponds to a trapping force of 194 ±
35 pNaccording to Faxen’s formula (see Method section E),which lies in the range predicted by theory in sectionIII. As a comparison, this force is one order of magni-tude larger than the maximum forces (20 pN) reported byKeloth et al . with optical tweezers and obtained withone order of magnitude less power (1 . . .After the tweezers were switched off, the cell did not dis-play any increase of fluorescence and remained at an in-tensity well under the dead cells found nearby (5 × to 10 × lower, see SI). This strongly supports that short-termdamages produced by the acoustical tweezers is minimal.It is however known that damages experienced by a cellcan lead to its death for hours afterwards . To assessthe long-term impact of cell manipulation using acous-tical tweezers, we performed a viability assay overnight.The MDA cells were seeded at 60 (%) confluence ratioin two glass devices with no surface treatment and leftto re-adhere for 5h. Nine cells located at different po-sitions in the two different microfluidic chambers wereexposed to the tweezers of acoustic vortex at maximumpower for 2 min each. An observation of the cells wasperformed after 19h (half the population doubling rateof MDA cells ) to compare their viability with a controlregion of the device (see Fig. 4A). No extra mortality wasobserved in the illuminated region (dead/live cell ratio of3%) compared to the statistics performed on the overalldevice (dead/live cell ratio of 5%). This likely indicatesthat the dead cells are depositing randomly and that the
400 um350 µm100 µm100 µm 100 µm100 µm
ABC DE
Areas withmanipulatedcells:Blue: cells alive Red: dead cellsInsonified cells
Cells viability after 19h
FIG. 4. A. Overview of the central part of the microfluidic device in which the viability experiments were performed. The cellsare stained using a viability kit and imaged at 360 nm and 535 nm excitation (460 nm and 617 nm emission). The cell nucleusare represented in blue, while the dead cells appear in red. The whole field of view contains 4581 cells (226 dead - 5%) whilethe region where manipulation took place contains 166 cells (5 dead - 3%). B-E Details of the 5 cells exposed to the acousticaltweezers for 2 min (4 others were exposed on another similar device). The green circle represents the first ring of the trap. tweezers do not provoke extra mortality. We also studiedin detail the fate of the nine illuminated individual cells(see Fig. 4B-E). All the cells exposed to the acoustic field(the green circle indicates the extension of the first ringof the vortex) and their immediate neighbours were aliveand showed no difference compared to the nearby cells.
V. CONCLUSION AND OUTLOOKS
In this work, cell harmless selective manipulation isdemonstrated through the capture and precise position-ing of individual cells amongst a collection in a standardmicroscopy environment. Both short-term and long-termviability of manipulated cells is evaluated, showing no im-pact on cells integrity. This opens widespread perspec-tives for biological applications wherein precise organiza-tion of cells or microorganisms is a requisite. In addi-tion, trapping force over wave intensity ratio two ordersof magnitude larger than the one obtained with opticaltweezers is reported with no deleterious effect such asphototoxicity. Further engineering optimization of thesetweezers to limit Joule heating will hence enable the ap-plication of stresses several orders of magnitude largerthan with optical tweezers without altering cells viabil-ity, a promising path for acoustic spectroscopy , celladhesion or cell mechano-transduction investiga-tion. In addition, new abilities could be progressivelyadded to these tweezers: The focused vortex structureused for selective particle trapping in this paper is alsoknown to exhibit 3D trapping capabilities . This func-tion was not investigated here owing to the confined na-ture of the microchamber but could closely follow this work. Synchronized vortices could also be used to assem-ble multiple particles, as recently suggested by Gong &Baudoin . This would enable the investigation of tissueengineering and envision 3D cell printing. Finally, themost thrilling and challenging perspective to this workmight be the future development of Spatial UltrasoundModulators (analogs to Spatial Light Modulator in op-tics), designed to manipulate and assemble many objectssimultaneously. While such a revolution is on the way forlarge particles manipulation in air , it would consti-tute a major breakthrough at the microscopic scale inliquids wherein the actuation frequencies are 3 ordersof magnitude larger. The present work hence consti-tutes the cornerstone towards widespread applications ofacoustical tweezers for biological applications. ∗ Corresponding author: [email protected];http://films-lab.univ-lille1.fr/michael † Corresponding author: [email protected] M. Baudoin and J.-L. Thomas, “Acoustical tweezers forparticle and fluid micromanipulation,” Annu. Rev. FluidMech. , 205–234 (2020). S.B.Q. Tran, P. Marmottant, and P. Thibault, “Fastacoustic tweezers for the two-dimensional manipulation ofindividual particles in microfluidic channels,” Appl. Phys.Lett. , 114103 (2012). X. Ding, S.-C. Lin, B. Kirali, H. Yue, S. Li, I.-K. Chiang,J. Shi, S. Benkovic, and T. J. Huang, “On-chip manipu-lation of single microparticles, cells, and organisms usingsurface acoustic waves,” Proc. Nat. Acad. Sci. USA ,11105–11109 (2012). A. Marzo, S.A. Seah, B.W. Drinkwater, D.R. Sahoo,B. Long, and S. Subramanian, “Holographic acoustic el-ements for manipulation of levitated objects,” Nat. Com-mun. , 8661 (2015). D. Baresch, J.-L. Thomas, and R. Marchiano, “Observa-tion of a single-beam gradient force acoustical trap for elas-tic particles: acoustical tweezers,” Phys. Rev. Lett. ,024301 (2016). K. Melde, A.G. Mark, T. Qiu, and P. Fischer, “Hologramsfor acoustis,” Nature , 518–522 (2016). A. Riaud, M. Baudoin, O. Bou Matar, L. Becerra, andJ.-L. Thomas, “Selective manipulation of microscopic par-ticles with precursos swirling rayleigh waves,” Phys. Rev.Appl. , 024007 (2017). M. Baudoin, J.-C. Gerbedoen, A. Riaud, O. Bou Matar,N. Smagin, and J.-L. Thomas, “Folding a focalized acous-tical vortex on a flat holographic transducer: miniaturizedselective acoustical tweezers,” Science Adv. , eaav1967(2019). R. Lirette, J. Mobely, and L. Zhang, “Ultrasonic extrac-tion and manipulation of droplets from a liquid-liquid in-terface with near fields acoustic tweezers,” Phys. Rev. App. , 061001 (2019). B.T. Hefner and P.L. Marston, “An acoustical helicoidalwave transducer with applications for the alignment of ul-trasonic and underwater systems,” J. Acoust. Soc. Am. , 3313–3316 (1999). P. Eifeng, J. Ing, Y. Annan, E. Thang, G.K. Eeler, N. Elly,M.C. Ruz, B. Enjamin, S.F. Reedman, L. Ih, and Y.L. In,“Optical tweezers system for live stem cell organizationat the single-cell level,” Biomed. Opt. Express , 771–779(2018). S.M. Block, D.F. Blair, and H.C. Berg, “Compliance ofbacterial flagella measured with optical tweezers,” Nature , 514 (1989). S. Suresh, “Biomechanics and biophysics of cancer cells,”Acta Biomater. , 413–438 (2007). D.H. Ou-Yang and M.-T. Wei, “Complex fluids: Probingmechanical properties of biological systems with opticaltweezers,” Annu. Rev. Phys. Chem. , 421–440 (2010). M.J. Aebersold, H. Dermutz, C. Forr, S. Weydert,G. Thompson-Steckel, J. Vrs, and L. Demk, “Brains on achip: Towards engineered neural networks,” TrAC-TrendsAn. Chem. , 60 – 69 (2016). G. Thalhammer, R. Steiger, S. Bernet, and M. Ritsch-Mart, “Optical macro-tweezers: trapping of highly motile micro-organisms,” J. Opt. , 044024 (2011). D. Ahmed, A. Ozcelik, N. Bojanala, N. Nama, A. Upad-hyay, Y. Chen, W. Hanna-Rose, and T.J. Huang, “Ro-tational manipulation of single cells and organisms usingacoustic waves,” Nat. Commun. , 11085 (2016). A. Ashkin, J.M. Dziedzic, J.E. Bjorkholm, and S. Chu,“Observation of a single-beam gradient force optical trapfor dielectric particles,” Optics letters , 288–290 (1986). D.G. Grier, “A revolution in optical manipulation,” Nature , 810 (2003). K.C. Neuman and S.M. Block, “Optical trapping,” Rev SciInstrum. , 2787–2809 (2004). F.H.C. Crick and A.F.W. Hugues, “The physical propertiesof cytoplasm,” Exp. Cell Res. , 37–80 (1950). S.B. Smith, L. Finzi, and C. Bustamante, “Direct mechan-ical measurements of the elasticity of single DNA moleculesby using magnetic beads,” Science , 1122–1126 (1992). T.R. Strick, J.-F. Allemand, D. Bensimon, A. Bensimon,and V. Croquette, “The elasticity of a single supercoileddna molecule,” Science , 1835–1837 (1996). K.C. Neuman, E.H. Chadd, G.F. Liou, K. Bergman, andS.M. Block, “Characterization of photodamage to Es-cherichia coli in optical traps,” Biophys. J. , 2856–2863(1999). Y. Liu, D.K. Cheng, G.J. Sonek, M.W. Berns, C.F. Chap-man, and B.F. Tromberg, “Evidence of localized cell heat-ing induced by infrared optical tweezers,” Biophys. J. ,2137–2144 (1995). Y. Liu, G.J. Sonek, M.W. Berns, and B.J. Tromberg, “As-sessing the effects of confinement by 1064-nm laser tweezersusing microflurometry,” Biophys. J. , 2158–2167 (1996). A. Blasquez, “Optical tweezers: Phototoxicity and thermalstress in cells and biomolecules,” Micromachines , 507(2019). A. Lenshof and T. Laurell, “Continuous separation of cellsand particles in microfluidic systems,” Chem. Soc. Rev. , 1203–1217 (2010). G. Sitters, D. Kamsa, G. Thalhammer, M. Ritsch-Marte,E.J.G. Peterman, and G.J.L. Wuite, “Acoustic force spec-troscopy,” Nat. Meth. , 47–52 (2015). D.J. Collins, B. Morahan, J. Garcia-Bustos, C. Doerig,M. Plebanski, and A. Neild, “Two-dimensional single-cellpatterning with one cell per well driven by surface acousticwaves,” Nat. Commun. , 8686 (2015). A. Ozcelik, J. Rufo, F. Guo, J. Li, P. Lata, and T.J.Huang, “Acoustic tweezers for the life science,” Nat. Meth. , 1021–1028 (2018). H. Bruus, “Acoustofluidics 7: The acoustic radiation forceon small particles,” Lab Chip , 1014–1021 (2012). D. Zhao, J.-L. Thomas, and R. Marchiano, “Computationof the radiation force exerted by the acoustic tweezers usingpressure field measurements,” J. Acoust. Soc. Am. ,1650–1660 (2019). F.S. Foster, C.J. Pavlin, K.A. Harasiewicz, D.A. Christo-pher, and D.H. Turnbull, “Advances in ultrasound biomi-croscopy,” Ultras. Med. & Biol. , 1–27 (2000). J. Hultstr¨om, O. Manneberg, K. Dopf, H.M. Hertz,H. Brismar, and M. Wiklund, “Proliferation and viabil-ity of adherent cells manipulated by standing-wave ultra-sound in a microfluidic chip,” Ultras. Med. Biol. , 145–151 (2007). M. Wiklund, “Acoustofluidics 12: Biocompatibility andcell viability in microfluidic acoustic resonators,” Lab Chip , 2018–2028 (2012). M.A. Burguillos, C. Magnusson, M. Nordin, A. Lenshof,P. Austsson, M.J. Hansson, E. Elmr, H. Lilja,P. Brundin, T. Laurell, and T. Deierborg, “Microchan-nel acoustophoresis does not impact survival or function ofmicroglia, leukocytes or tumor cells,” PloS ONE , e64233(2013). V. Marx, “Biophysics: using sound to move cells,” Nat.Meth. , 41–44 (2015). T.L. Szabo, “Ultrasound-induced bioeffects,” in
Diagnos-tic Ultrasound Imaging: Inside Out. , edited by AcadamicPress (Academic Press, 2014) Chap. 13. A. Keloth, O. Anderson, D. Risbridger, and L. Pater-son, “Single cell isolation using optical tweezers,” Micro-machines , 434 (2018). R.W. Boyle, “Ultrasonics,” Science Progress , 75–105(1928). G.T. Silva, J.G. Lopes, J.P. Leao-Neto, K. Nichols, andB. Drinkwater, “Particle patterning by ultrasonic stand-ing waves in a rectangular cavity,” Phys. Rev. Appl. ,054044 (2019). J. Lee, S.-Y. Teh, A. Lee, H.H. Kim, C. Lee, and K.K.Shung, “Single beam acoustic trapping,” Appl. Phys. Lett. , 073701 (2009). L.P. Gor’kov, “On the forces acting on a small particle inan acoustical field in an ideal fluid,” Sov. Phys. Dokl. ,773 (1962). D. Baresch, J.-L. Thomas, and R. Marchiano, “Three-dimensional acoustic radiation force on an arbitrarily lo-cated elastic sphere,” J. Acoust. Soc. Am. , 25 (2013). D. Baresch, J.-L. Thomas, and R. Marchiano, “Sphericalvortex beams of high radial degree for enhanced single-beam tweezers,” J. Appl. Phys. , 184901 (2013). C.R.P. Courtney, C.E.M. Demore, H. Wu, A. Grinenko,P.D. Wilcox, S. Cochran, and B.W. Drinkwater, “Inde-pendent trapping and manipulation of microparticles us-ing dexterous acoustic tweezers,” Appl. Phys. Lett. , 154103 (2014). M. Settnes and H. Bruus, “Forces acting on a small particlein an acoustic field in a viscous fluid,” Phys. Rev. E ,016327 (2012). P. Augustsson, J.T. Karlsen, H.-W. Su, H. Bruus, andJ. Vlodman, “Iso-acoustic focusing of cells for size-sensitiveacousto-mechanical phenotyping,” Nat. Commun. , 11556(2016). O.A. Sapozhnikov and M.R. Bailey, “Radiation force of anarbitrary acoustic beam on an elastic sphere in a fluid,” J.Acoust. Soc. Am. , 661–676 (2013). J. Happel and H.. Brenner,
Low Reynolds number hy-drodynamics with special applications to particulate media (Springer., 1965). M.-P. Rols, “Parameters affecting cell viability followingelectroporation in vitro,” Handbook of Electroporation ,1449–1465 (2017). M.B. Zeigler and D.T. Chiu, “Laser selection significantlyaffects cell viability following single-cell nanosurgery,” Pho-tochem Photobiol. , 1218?1224 (2009). ATCC, “Thawing, propagation and cryopreservation ofnci-pbcf-htb26 (mda-mb-231) breast adenocarcinoma,”Bioressource Core Facility (2012). J.T. Parsons, A.R. Horwitz, and M.A. Schwartz, “Celladhesion: integrating cytoskeletal dynamics and cellulartension.” Nat. Rev. Mol. Cell Biol. , 633–643 (2010). Z. Gong and M. Baudoin, “Particle assembly with synchro-nized acoustical tweezers,” Phys. Rev. Appl. , 024045(2019). D. Foresti and D. Poulikakos, “Acoustophoretic contactlesselevation, orbital transport and spinning of matter in air,”Phys. Rev. Lett. , 024301 (2014). A. Marzo and B.W. Drinkwater, “Holographic acoustictweezers,” Proc. Nat. Ac. Sci. , 84–89 (2019). R. Hirayama, D.M. Plasencia, N. Masuda, and S. Subra-manian, “A volumetric display for visual, tactile and audiopresentation using acoustic trapping,” Nature575