This document reviews acoustic trapping and manipulation of living cells. It discusses how acoustic manipulation works by trapping cells in pressure gradients using ultrasonic standing waves. The ability to non-invasively trap and manipulate live cells could enable applications like selective drug delivery and studying cell-cell interactions. Various acoustic manipulation techniques are described, including using opposing transducers to generate a standing wave for trapping cells. Characterizing the effects on cell viability and measuring acoustic forces are important. The project aims to design a sterilizable device that can optically image cells while acoustically trapping them. Initial tests trapping flour suggest the approach may work for cells.

1. A Brief Review of Acoustic Trapping and Manipulation of Living cells
Frederick A. O. White
(Dated: December 3, 2014)
The development of centimetre and micrometre scale acoustic manipulation systems has led to increasing
interest in their possible applications in biophysics. This review investigates the current state of the art in
acoustic manipulation, its challenges and potential applications to the study of live cells. The ability to trap
and manipulate live cells raises the possibility of selective labelling and drug delivery to subsets of cells in a
controlled environment. Various approaches to manipulating cells are considered and discussed.
INTRODUCTION
Acoustic trapping was first observed in 1868 during
Kundt’s tube experiments, where cork dust aligned with the
standing wave in an air-filled resonant cylinder[1]. The first
theoretical description of the acoustic radiation force for in-
compressible spheres was produced in 1934 by King[2]. This
was extended to compressible particles in 1955 by Yosioka
and Kawasima[3] and finally generalised for viscid fluids in
1962 by Gor’kov, whose derivation is most commonly used
today[4].
Acoustic manipulation traps cells by the pressure gradient
produced in a non-uniform acoustic field. Interest in acoustic
manipulation for biophysics applications has grown rapidly
over the last decade. Acoustic manipulation enables non-
invasive, non-contact trapping of cells and particles without
the need to remove them from their culture[5]. This can ben-
efit the study of cells by allowing the study of groups of cells
in a controlled environment over longer periods than possible
if cells can move freely. Acoustic manipulation is less likely
to damage cells than mechanical techniques and allows trap-
ping without affecting the environment around the cell, mak-
ing it suitable for delicate cell cultures and precise studies[6].
Acoustic manipulation has advantages over other non-contact
methods being cheap, easy to integrate into microfluid sys-
tems and maintaining cell viability[6]. The only requirement
of cells for acoustic trapping is a density and compressibility
contrast with the cell-carrying medium, so it is applicable to
a wide range of cells and particles[7, 8]. One disadvantage
is the relatively low precision and poor single-cell control, al-
though recent work has begun to mitigate these issues using
higher order Bessel functions while manipulating µm scale
polystyrene beads[9]. The main focus of this review, how-
ever, will be the application of acoustic manipulation to living
cells, and its success and future potential for cell studies.
THEORY
Acoustic manipulation uses the primary radiation force
(PRF). This arises when an ultrasonic standing wave inter-
acts by scattering off a particle or cell with an acoustic con-
trast to the fluid medium. The PRF moves particles to a point
of either maximum or minimum acoustic potential depending
on this contrast[5]. The standard approach, originally due to
Gor’kov[4], is to write the PRF as the gradient of the acous-
tic potential. A full derivation is available in Bruus’ 2007
paper[10] the result of which is:
Frad
= − Urad
(1a)
Urad
=
4π
3
a3
f1
1
2
κ0 p2
1 − f2
3
4
ρ0 v2
1 (1b)
where v2
1 and p2
1 are the time-averaged incoming velocity
and pressure fields squared, κ0 and ρ0 are the compressibility
and density of the fluid and the subscript p in Eq.2 denotes
the same quantities for the spherical particle radius a. The
quantities f1 and f2 are dimensionless scattering coefficients
defined by the value of the compressibility and density of the
particle compared to that of the surrounding fluid.
f1(˜κ) = 1 − ¯κ with ¯κ =
κp
κ0
(2a)
f2(˜ρ) =
2(˜ρ − 1)
2˜ρ + 1
with ˜ρ =
ρp
ρ0
(2b)
The form of these coefficients means that most cells will
move to the minima in the potential since their density tends
to be higher and their compressibility lower than the surround-
ing medium[5]. Once the first cells are entrapped, secondary
forces gather nearby cells into a cluster centred on the nodal
point. It is significant that the acoustic force experienced
by a cell scales with volume, not radius, so smaller particles
are harder to manipulate as the Stokes’ drag they experience
scales with radius and so falls off more slowly than the applied
trapping force.
ALTERNATIVES TO ACOUSTIC MANIPULATION
There are several alternative approaches to non-contact cell
handling besides acoustic manipulation. Optical tweezers trap
cells at the focal point of a laser beam. This has the ad-
vantage of extreme precision, on the scale of nanometres.
However, the laser can deposit significant energy in cells
and the equipment is difficult to integrate with cell-culture
containers[6, 11]. Additionally, optical tweezers are limited
to <10µm cell diameters, whereas acoustic manipulation can

2. 2
reach the range of ∼100µm[11]. The ability of optical tweez-
ers to manipulate single cells has led to interest in combining
optical tweezers and acoustic manipulation to use the best fea-
tures of both: acoustic manipulation to handle large groups of
cells and operate over relatively large areas but with the pre-
cision of optical tweezers for single cells[12].
Magnetic trapping and electrophoresis use non-uniform
magnetic and electric fields respectively. Magnetic trapping is
limited in its applicability to live-cell studies by the need for
either naturally magnetic cells or doped cells. Electrophore-
sis provides better precision than acoustic manipulation but
requires strong gradients in the field, which limits the work-
ing distance as the electrodes must be closely spaced. There is
also the risk of Joule heating in fluid media which can damage
cells[13].
APPLICATIONS OF ACOUSTIC MANIPULATION
The ability to immobilise cells is useful as it allows the
study of particular groups of cells in a strictly controlled en-
vironment, since a well designed acoustic device should not
significantly affect the medium or cells. This makes it suit-
able for for a wide range of biophysics applications.
Acoustic manipulation has been applied in high-
resolution cell response studies and to characterise cell–cell
interactions[14]. Other applications include reducing the
time needed to create 3D cell clusters and enhance bioassays,
which may have applications in tissue engineering[11].
Ultrasound in the MHz range has been applied for filtration
and cell gathering since the 90s[15, 16]. Initially filtration
was achieved by agglomerating cells using acoustic ma-
nipulation and allowing them to settle before drawing off
clear fluid. More recently work on micrometre scales has
achieved in-flow filtering and cell washing. Working with
flowing media allows cell environments to be maintained
during processing[5, 17]. A recent application of acoustic
manipulation is in the study of non-adherent cells, where the
ability to group cells that do not naturally cluster or grow on
surface cultures is of great benefit[14].
DESIGN OF DEVICES
The standing wave field required for acoustic manipulation
can be produced in several ways. Ultrasound waves are al-
most always produced using piezoelectric ceramics[7]. For
all devices the acoustic impedance of various layers must be
considered to either transmit acoustic energy or reflect it as
necessary.
The most common approach is a multilayer resonant sys-
tem, consisting of stacked layers, from bottom to top: trans-
ducer, coupling layer, fluid, and a reflecting layer. The thick-
ness of the layers must be tuned carefully to achieve reso-
nance. This dependence on geometry limits resonant systems’
manipulation capacity since the nodal positions of the acous-
tic field are fixed. Other approaches include using focused
ultrasound beams (first developed by Wu in 1991 [18]) in a
method similar to optical tweezers, or using a linear array of
transducers opposite a reflector. Linear arrays have been ap-
plied successfully in 300µm channels by Demore et al.[19].
The final approach, and the one applied in this project, is to
produce a standing wave using an opposed pair of transducers
emitting counter-propagating travelling waves. This has the
advantage of being unaffected by changes in the resonant fre-
quency of the chamber due to large numbers of particles in the
fluid[20]. One challenge is to eliminate reflection at the trans-
ducers, to avoid production of a resonant wave in addition to
the desired standing wave. Arbitrary positioning of the nodal
points by adjustment of the relative phases of the waves and
rapid variation of the field is possible even in a chamber sev-
eral wavelengths long. This is advantageous for fine control of
particles and also allows a larger working volume in compar-
ison to microfluidic resonant systems. While opposing trans-
ducers allow arbitrary nodal positioning, the traps are always
at half-wavelength separation and any phase variation affects
all traps simultaneously[21]. An early example of an opposing
transducer system was developed by Kozuka[22], with recent
work by Courtney et al. demonstrating manipulation in one
and two dimensions[21, 23]. Manipulation in two dimensions
is achieved by using two opposed pairs emitting at 90◦
to each
other, producing a grid-like series of nodes. One limiting fac-
tor in this technique is the frequency and impedance matching
of the opposed transducers. The closer in frequency the two
travelling waves are, the greater the amplitude of the stand-
ing wave created and the stronger the trapping. Impedance
matching ensures similar mechanical responses under load
from each transducer, so their resonances shift together[24].
Grinenko et al. have demonstrated the possibility of work-
ing with unmatched transducers by the application of acoustic
absorbers behind the transducers[25].
Acoustic streaming presents a problem for all manipula-
tion and trapping. This is caused by the bulk fluid absorbing
acoustic energy, leading to flow, which can force cells out of
the area of interest via drag forces and cause unwanted stress
on cells[26]. There are three forms of streaming: Eckart,
Schlichting and Rayleigh. Eckart streaming is the largest in
magnitude but is strongly influenced by the depth of fluid
and can be reduced significantly by working in shallower
fluids[27].
CHARACTERISING A DEVICE – EFFECTS ON CELL
VIABILITY AND MANIPULATION CAPABILITIES
Having constructed a functioning device for acoustic ma-
nipulation, it is necessary to characterise its capabilities and
impact on cells. The first step in any cell study is to main-
tain viability. There is large body of evidence that medical
ultrasound at low intensity has no significant impact on hu-
man tissue[28, 29]. While this is true for bulk tissues it may

3. 3
not hold true for individual cells or different cell types, so
each case must be considered individually. In general the
damaging effects of ultrasound are considered as thermal or
non-thermal. Thermal effects are problematic since most cells
have a suitable temperature range for proliferation of a few
degrees centigrade, thus heating can easily push the culture
out of this range. Fortunately thermal absorption by water in
the range of 1–10MHz, most commonly used for ultrasonic
acoustic trapping, is low. In the case of small volumes, di-
rect heat transfer from transducers can present issues, but this
can be mitigated with careful cooling of the transducers [30].
Temperature in acoustic devices can be tracked via fluores-
cence studies using Rhodamine B[11]. Using Rhodamine re-
moves the need for a physical thermocouple which would dis-
turb the acoustic field.
Non-thermal effects include stresses applied to cells by the
acoustic radiation force, streaming (see previous page) and
cavitation. Cavitation is generally negligible for the MHz fre-
quencies used in acoustic manipulation; it also requires nucle-
ating sites for bubble formation, so can be mitigated by use of
degassed water with few microscopic bubbles present[31].
Despite the potential for adverse effects, several studies
have demonstrated the viability of cells held in ultrasound
traps. Bazou et al. tested embryonic stem cells[26, 32], Hul-
str¨om COS-7 cells[33], Evander et al. yeast cells[11] and
Haake et al. HeLa cells[34]. Each group was working with
their own device, demonstrating that cells remain viable un-
der a variety of acoustic conditions. The simplest method for
quantifying the impact of trapping on cell viability is through
observations of cell membrane integrity using an optical mi-
croscope or cell-counting device. Several dyes exist which in-
crease the visibilty of damaged cells by either tracking active
metabolic processes or only penetrating ruptured cells[30].
A recently developed method for direct measurement of the
force uses optical tweezers. Particles are held in the optical
tweezers at a known position with known force, since optical
trapping has well–established parameters. The acoustic power
is increased until the particle escapes the optical trap. An ad-
vantage of this route is that it makes no assumptions about the
fluid or pressure field. Another route to measuring forces is
to apply particle image velocimetry as particles move towards
the nodal points in a field[24].
PROJECT PROGRESS
The aim of the project is to design and construct a
centimetre-scale ultrasound trap which maintains cell viabil-
ity, allows optical access for live imaging and can be sterilised
for reuse.
Initial design possibilities were suggested by Dr Berry and
Dr Barnes in the form of a centimetre-scale device developed
in the Biosciences group. This device worked with two pairs
of opposed piezoelectric transducers in a 3D printed mount
which is submerged in the cell–culture medium. This led to
difficulties with sterilisation and construction of the power
FIG. 1: The original device showing the circular mount holding the
transducers – this is then inserted into a petri–dish containing the
cell–culture.
supplies, since they are fed around the petri–dish cover. It
was also reported that the device struggled to maintain cell vi-
ability. However, as an initial device for developing skills and
testing construction methods it is proving useful.
Design of a new device and preliminary testing with the
original were carried out in tandem. Whatever final design is
chosen the piezoelectric crystals must be characterised care-
fully for optimal trapping. We tested the original device using
a Wayne-Kerr 6500b impedance analyser to locate the reso-
nances of the transducers. Using the resonant frequency is
vital to achieve maximum conversion of electrical to mechan-
ical power. Having characterised the device, we carried out
initial tests using flour as a cheap substitute for micro-beads.
The result was successful alignment of flour grains parallel to
the driven pair of transducers using a frequency of 6.915MHz,
as shown in Fig. 2. The larger clumps of flour were beyond
the capacity of the device to trap but the smaller particles are
seen aligned. These tests allowed us to gain experience with
the Olympus SZX-16 microscope being used for optical ob-
servations.
When developing a new device, the first decision was
whether to locate the transducers within the fluid medium,
for best transmission of acoustic energy, or outside the fluid
chamber for ease of sterilisation. It was decided that sepa-
rating the fluid and transducers would make sterilisation sim-

4. 4
FIG. 2: Flour-water suspension aligned using the initial device.
The thin lines of flour formed when the transducers were driven at
6.915MHz and 7.5 Vpp while the larger clumps were unaffected.
pler. Our first test–bed device, shown in Fig. 4 is based on
the work of Scholz et al., with a smaller fluid chamber[20].
It uses two opposed transducers and a central, square fluid
chamber mounted on a standard microscope slide using sili-
cone glue. It is hoped future iterations will provide phase con-
trol of individual transducers for 1D manipulation and even-
tually introduce an additional pair of transducers for 2D ma-
nipulation. The transducers will be held in position either us-
ing 3D printed x-shaped mounts, as in Fig. 4, or small com-
pression springs. Initial working sketches of a second gen-
eration device are shown in Fig.5 The first generation de-
vice has been produced in polymethyl methacrylate (PMMA)
acrylic and glass to test the acoustic properties of both mate-
rials. This involved producing 3D models of the device for
laser and water cutting. The water cutting was carried out in
the university workshops as it required specialist equipment.
The laser cutting took place on the student-accessible device
FIG. 3: The two initial devices - the upper is cut in glass, the lower
in acrylic.
FIG. 4: Schematic diagram of the new device, all dimensions are
in mm. The position of one mounted transducer is shown in blue,
the 3D printed mount in orange. The finished device will have a
symmetrical pair.
FIG. 5: Proposed layout for a device with two opposed pairs of trans-
ducers - the dimensions are currently the same as for the first iteration
device.
in the engineering department. With the help of Philip Bassin-
dale we learned to program this ourselves and will be able to
produce future designs without assistance. The glass device
has grooves cut for power supplies and we will add these to
the acrylic device before testing. With the transducers outside
the fluid chamber a coupling medium will be needed between
the transducer and the container. Initial tests will use commer-
cially available EE1295 ultrasound gel, since this is designed
to impedance match water–dominated tissue and our fluid will
be water based.
We will be working with human epithelial and corneal cells
which require containment level 1 procedures. The necessary
training is being provided by Dr Berry. We hope to be able
to produce our own cell cultures and suspensions of desired
concentrations for cell studies. Initial tests will be carried out
using fixed, dyed cells of the same type as will be used live
later in the project.
CONCLUSION
The potential of acoustic manipulation of live cells has been
well–established. Opposing transducer methods have been
used relatively little in biophysics but engineering applications
have shown them to have the precision and power required for
handling live cells. Non-contact manipulation of cells in this
way has the potential to provide targeted signalling molecule

5. 5
delivery to subgroups of cells in the same environment for di-
rect comparison.
ACKNOWLEDGEMENTS
Dr Barnes and Dr Berry for their support in planning and
initiating the project. Thanks must also go to John Rowden
and Philip Bassindale for their assistance in producing the
glass and laser-cut devices.
[1] A. Kundt. Acoustic experiments. Phil. Mag., 4:41–48, 1868.
[2] L. V. King. On the acoustic radiation pressure on spheres. Pro-
ceedings of the Royal Society of London. Series A - Mathemat-
ical and Physical Sciences, 147(861):212–240, 1934.
[3] K. Yosioka and Y. Kawasima. Acoustic radiation pressure on
a compressible sphere. Acta Acustica united with Acustica,
5(3):167–173, 1955.
[4] L.P. Gor’kov. On the Forces Acting on a Small Particle in an
Acoustical Field in an Ideal Fluid. Soviet Physics Doklady,
6:773, March 1962.
[5] M. Evander and J. Nilsson. Acoustofluidics 20: Applications in
acoustic trapping. Lab Chip, 12:4667–4676, 2012.
[6] Y. Qiu et al. Acoustic devices for particle and cell manipulation
and sensing. Sensors, 14:14806–14838, 2014.
[7] T. Laurell, F. Petersson, and A. Nilsson. Chip integrated strate-
gies for acoustic separation and manipulation of cells and par-
ticles. Chem. Soc. Rev., 36:492–506, 2007.
[8] D. Bazou et al. Gene expression analysis of mouse embryonic
stem cells following levitation in an ultrasound standing wave
trap. Ultrasound in Medicine & Biology, 37(2):321 – 330, 2011.
[9] C. R. P. Courtney et al. Independent trapping and manipulation
of microparticles using dexterous acoustic tweezers. Applied
Physics Letters, 104(15), 2014.
[10] H. Bruus. Acoustofluidics 7: The acoustic radiation force on
small particles. Lab Chip, 12:1014–1021, 2012.
[11] M. Evander et al. Noninvasive acoustic cell trapping in a mi-
crofluidic perfusion system for online bioassays. Analytical
Chemistry, 79(7):2984–2991, 2007. PMID: 17313183.
[12] G. Thalhammer et al. Combined acoustic and optical trapping.
Biomed. Opt. Express, 2(10):2859–2870, Oct 2011.
[13] C. Duschl et al. Versatile chip-based tool for the controlled
manipulation of microparticles in biology using high frequency
electromagnetic fields. In H. Andersson and A. Berg, editors,
Lab-on-Chips for Cellomics, pages 83–122. Springer Nether-
lands, 2004.
[14] J. Nilsson, M. Evander, B. Hammarstrm, and T. Laurell. Review
of cell and particle trapping in microfluidic systems. Analytica
Chimica Acta, 649(2):141 – 157, 2009.
[15] W. T. Coakley. Ultrasonic separations in analytical biotechnol-
ogy. Trends in Biotechnology, 15(12):506 – 511, 1997.
[16] W.T. Coakley, J.J. Hawkes, M.A. Sobanski, C.M. Cousins, and
J. Spengler. Analytical scale ultrasonic standing wave manip-
ulation of cells and microparticles. Ultrasonics, 38(18):638 –
641, 2000.
[17] J. J. Hawkes, R. W. Barber, D. R. Emerson, and W. T. Coakley.
Continuous cell washing and mixing driven by an ultrasound
standing wave within a microfluidic channel. Lab Chip, 4:446–
452, 2004.
[18] J. Wu. Acoustical tweezers. The Journal of the Acoustical So-
ciety of America, 89(5), 1991.
[19] C. Demore et al. Transducer arrays for ultrasonic particle ma-
nipulation. In Ultrasonics Symposium (IUS), 2010 IEEE, pages
412–415, Oct 2010.
[20] M. S. Scholz, B. W. Drinkwater, and R. S. Trask. Ultrasonic
assembly of short fibre reinforced composites. In Ultrason-
ics Symposium (IUS), 2014 IEEE International, pages 369–372,
Sept 2014.
[21] C. R. P. Courtney, C.-K. Ong, B. W. Drinkwater, A. L. Bernas-
sau, P. D. Wilcox, and Cumming D.R.S. Manipulation of par-
ticles in two dimensions using phase controllable ultrasonic
standing waves. Proc. R. Soc. A, 468:337–260, 2012.
[22] T. Kozuka et al. Control of position of a particle using a stand-
ing wave field generated by crossing sound beams. In Ultra-
sonics Symposium, 1998. Proceedings., 1998 IEEE, volume 1,
pages 657–660 vol.1, 1998.
[23] C. R. P. Courtney, C.-K. Ong, B. W. Drinkwater, P. D. Wilcox,
C. Demore, S. Cochran, P. Glynne-Jones, and M. Hill. Ma-
nipulation of microparticles using phase-controllable ultrasonic
standing waves. The Journal of the Acoustical Society of Amer-
ica, 128(4), 2010.
[24] P. G. Bassindale, D. B. Phillips, A. C. Barnes, and B. W.
Drinkwater. Measurements of the force fields within an acous-
tic standing wave using holographic optical tweezers. Applied
Physics Letters, 104(16):–, 2014.
[25] A. Grinenko et al. Efficient counter-propagating wave acoustic
micro-particle manipulation. Applied Physics Letters, 101(23),
2012.
[26] D. Bazou, A. K Larisa, and W. T. Coakley. Physical enviroment
of 2-d animal cell aggregates formed in a short pathlength ultra-
sound standing wave trap. Ultrasound in Medicine & Biology,
31(3):423 – 430, 2005.
[27] A. L. Bernassau et al. Controlling acoustic streaming in an
ultrasonic heptagonal tweezers with application to cell manipu-
lation. Ultrasonics, 54(1):268 – 274, 2014.
[28] M. W. Miller et al. Hyperthermic teratogenicity, thermal dose
and diagnostic ultrasound during pregnancy: implications of
new standards on tissue heating. International Journal of Hy-
perthermia, 18(5):361–384, 2002.
[29] M. C. Ziskin et al. Current status of research on biophysical ef-
fects of ultrasound. Ultrasound Med. Biol., 20:205–218, 1994.
[30] M. Wiklund. Acoustofluidics 12: Biocompatibility and cell vi-
ability in microfluidic acoustic resonators. Lab Chip, 12:2018–
2028, 2012.
[31] W. L. Nyborg. Biological effects of ultrasound: Development
of safety guidelines. part ii: General review. Ultrasound in Med.
& Biol., 27(3):301–333, 2001.
[32] D. Bazou, W. T. Coakley, A. J. Hayes, and S.K. Jackson. Long-
term viability and proliferation of alginate-encapsulated 3-d
hepg2 aggregates formed in an ultrasoun trap. Toxicology in
Vitro, 22(5):1321–31, 2008.
[33] J. Hulstr¨om et al. Proliferation and viability of adherent cells
manipulated by standing-wave ultrasound in a microfluidic
chip. Ultrasound in Med. and Biol., 33(1):145–151, 2007.
[34] A. Haake et al. Manipulation of cells using an ultrasonic pres-
sure field. Ultrasound in medicine & biology, 31(6):857–864,
2005.