Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves

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Received 15 Jan 2015 | Accepted 17 Sep 2015 | Published 2 Nov 2015 DOI: 10.1038/ncomms9686 OPEN
## Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves

David J. Collins 1, Belinda Morahan 2, Jose Garcia-Bustos 2, Christian Doerig 2, Magdalena Plebanski 3,4

& Adrian Neild 1

In single-cell analysis, cellular activity and parameters are assayed on an individual, rather

than population-average basis. Essential to observing the activity of these cells over time is

the ability to trap, pattern and retain them, for which previous single-cell-patterning work has

principally made use of mechanical methods. While successful as a long-term cell-patterning

strategy, these devices remain essentially single use. Here we introduce a new method for the

patterning of multiple spatially separated single particles and cells using high-frequency

acoustic fields with one cell per acoustic well. We characterize and demonstrate patterning

for both a range of particle sizes and the capture and patterning of cells, including human

lymphocytes and red blood cells infected by the malarial parasite Plasmodium falciparum. This

ability is made possible by a hitherto unexplored regime where the acoustic wavelength is on

the same order as the cell dimensions.

1 Department of Mechanical and Aerospace Engineering, Monash University, Clayton, Victoria 3800, Australia. 2 Department of Microbiology, Infection and
Immunity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. 3 Department of Immunology, Alfred
Hospital Precinct, Monash University, Melbourne, Victoria 3004, Australia. 4 Therapeutics and Regenerative Division, Monash Institute of Medical
Engineering, MIME, Monash University, Clayton, Victoria 3800, Australia. Correspondence and requests for materials should be addressed to A.N.
(email: [adrian.neild@monash.edu).](mailto:adrian.neild@monash.edu)

rapping and patterning of cells is fundamental for purposes
as varied as bioprinting 1,2, cell–cell interaction studies 3,
drug development 4 and single-cell analysis, a growing field
# T
of interest where heterogeneous cellular parameters are assessed
on the basis of individual cells and their response to their local
environment 5,6 . In cell studies, for example, constraining the
location of cells according to defined positions permits the
prolonged visualization of individual cellular development—
potentially in the presence of modifying reagents—allowing the
observation of phenomena that are averaged out in bulk cell
culture. Restricting cellular patterns to two dimensions further
aids in the visualization and study of individual cells, where the
en masse trapping of 100–1,000 s of cells allows single-cell
analysis on the scale of large populations 7,8 .

Microfluidic methods are a highly effective avenue for the
patterning of single cells, where the dimensions of force gradients
or physical features are, by necessity, on the same scale as
individual cells (B5–20 mm). Importantly, the distinction must
be made between microfluidic methods that allow patterning of
aggregates of cells or particles and those that enable this for
individual ones; although patterning of cellular aggregates is

Figure 1 | The design concept and example device used for OCPW
patterning. (a) A OCPW device is comprised of a microfluidic chamber
bonded to a piezoelectric substrate onto which interdigital transducers
(IDTs) are patterned to generate four sets of intersecting surface acoustic
waves (SAWs) that produce a high-frequency acoustic field in a coupled
body of liquid. This field, represented by the 2D wavefield in b and c traps
individual particles at nodal positions. (d) Here the chamber of the finished
device is filled with blue dye for visualization, with an Australian $2 coin for
scale. Adapted from ref. 70.

useful for many applications, it is only through the spatial
isolation of individual cells and the optical access that it affords
that single-cell analysis is possible. A number of microfluidic
techniques employ either hydrodynamic/mechanical methodologies or active forces to capture and pattern individual cells.
Hydrodynamic methods serve to passively steer individual cells in
a continuous flow to micro-patterned mechanical structures that
spatially exclude more than a defined number of cells 9–13 .
A major limitation of the mechanical trapping approach is that
these devices are mostly single-use; when a cell is captured for a
sufficient time it will adhere to the channel features and remain
trapped. While this is sufficient for many long-term cell culture
studies, for other applications such as the trapping and analysis of
rare cells it is desirable to dictate both the time and duration of
capture in addition to the location of cell trapping. A number
of active techniques have been used for particle and cell
manipulation and patterning, including optical 14,15, magnetic 16,
electrical 17 and acoustic 18–32 forces, although these differ in
their suitability to the patterning of individual, isolated cells.
High-frequency acoustic forces—where periodically fluctuating
pressure conditions result in time-averaged forces that push
suspended matter towards acoustic nodes/antinodes—are
generally biocompatible and have demonstrated potential for
long-term cell observation 22 . This avoids problems such as the
photobleaching of fluorescent enzymes and local heat stress
associated with optical trapping, or the induction of strong
electrical fields that can harm long-term cell viability in the case
of dielectrophoretic forces.

Although acoustic fields have demonstrated patterning of
particles and cells, for the most part the patterned cells form
aggregates, rather than spatially isolated individual cells 19,33 . In
this case it is still possible for a single cell to be individually
trapped, although this is the outcome of using a low initial sample
concentration, ultimately preventing the formation of relatively
dense patterns otherwise available in hydrodynamic patterning
methods 22,29 . There is, however, nothing about an acoustic field
that inherently prevents the patterning of individual cells. By
understanding the relevant forces in a high-density acoustic
pattern and by imposing an acoustic field with a smaller
wavelength than previously utilized, there is nothing to prevent
the patterning of single cells in individual minimum-force
locations. This physical regime, in which the particle or cell
diameter D approaches the acoustic wavelength l, has not been
explored to date.

Here, we utilize surface acoustic waves (SAWs) at high
frequency to create a two-dimensional (2D) acoustic force field
with an inter-nodal spacing of the same order as the patterned
cell dimensions. We hypothesize and experimentally verify that
the patterning of spatially isolated cell patterns is feasible within a
relatively narrow band of particle diameters as a ratio of the
acoustic wavelength, a result of the combined and competing
effects of the acoustic field and inter-particle interactions. When
an acoustic field within this range is generated, both particles and
cells are patterned in the 2D array of force potential minima with
one cell per acoustic well (OCPW). Figure 1 shows schematics
and a picture of the device that generates the acoustic field that is
used to pattern particles and cells.

Results
Device principles and design. We generate a SAW by applying
an a.c. voltage across a series of interdigital transducers (IDTs)
that are patterned on a piezoelectric lithium niobate substrate; as
with any piezoelectric material, a spatial gradient in electrical
potential results in a mechanical displacement, which continues
to propagate across the surface of that substrate—much as ripples

in a pond—until dissipated by relaxation in the material itself or
through a secondary material that the SAW couples into. SAW is
a term describing a number of wavemodes whose displacements
are concentrated at the substrate surface. SAW is uniquely suited
to microfluidic manipulation of cells, with the capacity to locally
generate an acoustic field of arbitrary aperture at a range of high
frequencies (B0.01–10 GHz) and subsequently small wavelengths
(B0.4–400 mm) 34,35 . Rayleigh-mode SAW, where the primary
displacement in a single-material substrate is orthogonal to the
surface and energy is transferred vertically into a fluid placed on
top, is the most commonly used SAW mode for microfluidics
applications 36–38 . Moreover, a SAW device is inherently planar,
and is therefore simple to combine with other on-chip
microfluidic features.

Applying the resonant frequency across the IDT electrodes so
that the mechanical displacements from a finger pair, the
minimum periodic unit of the IDT structure, reinforce one
another results in a high-displacement SAW. The resonant
frequency is given by f ¼ cs/l, where cs is the speed of sound in the
substrate and l is the acoustic wavelength as determined by the
spacing between adjacent IDT finger pairs. Despite the small
surface displacements typical of MHz–GHz SAW, typically on the
order of 0.1–10 nm, the resulting surface velocities are up to
1–10 ms � 1, which drive up to MPa-order standing-wave
pressures that can be used to capture particles and cells 39 . In
addition, as a result of the surface-bound nature of the
displacement, acoustic energy couples efficiently from the
substrate surface to a fluid in contact with it. A standing wave
in the horizontal plane of the fluid, necessary to trap particles, is
induced through the intersection of two counter-propagating
waves on the substrate surface. The addition of a channel roof in
the path of this wave also results in a partial reflection of the
propagating wave in the fluid, thus yielding a partial standing
wave in the vertical plane that acts to constrain the motion of
particles in this direction as well.

As the IDT spacing determines acoustic field’s wavelength,
different devices are used to access a range of OCPW-trapping
regimes for multiple particle sizes, with 15, 18, 21, 23, 25 and
36 mm wavelengths, in addition to a wide-spectrum chirped
device whose finger-pair wavelength and spacing varies from 14
to 60 mm, meaning that wavelengths in between these values can
be accessed. See the Methods section for device fabrication details.
A completed device is shown in Fig. 1d.

Acoustic forces. In this section, we consider the forces experienced by a particle in a 2D acoustic field. We find that successful
OCPW capture is not simply the imposition of a sufficiently small
wavelength, below which single particles are patterned, but rather
a relatively narrow band where the forces separating particles are
dominant over those attracting them together. This interplay
between these opposing forces, an understanding of which is
necessary for single-particle patterning, has not been examined
previously. Broadly put, a particle undergoes migration as a result
of a time-averaged force acting on that particle when subject to an
externally imposed acoustic field 40 . In a standing wave, this
acoustic radiation force acts to move particles along a potential
gradient towards the standing-wave node or antinode, depending
on the respective densities and compressibilities of the particle
and surrounding media; most types of cells and particles
suspended in water, though, will migrate towards standingwave nodal positions. However, in a system in which multiple
particles are trapped and patterned simultaneously, each particle
cannot be considered in isolation as they will also have an effect
on the surrounding acoustic field. Here, the incoming wave, while
driving particles towards the nodal positions, will also reflect and

thus scatter a portion of the acoustic energy in the particle’s
vicinity, leading to secondary inter-particle forces (so-called
Bjerknes forces) 41,42, which may either be repulsive or attractive,
though are more often attractive than repulsive for the typical
media and particles/cells that are commonly used.

The regime used for OCPW trapping, with particle dimensions
D approaching that of l, should be described in its relation to
previous acoustic trapping work. In Fig. 2, it can be seen that
substantially larger vales of D/l and smaller absolute values of l,
to account for the small size of cells, are necessary for OCPW
patterning. Through the imposition of particles at higher D/l,
only single particles can inhabit an individual nodal position due
to steric considerations, in contrast to the previously demonstrated cases where particle aggregates form at these locations.
The horizontal axis l � 1, underscores the substantially higher
frequencies (on the order of f \100 MHz, where fpl � 1 ) than
typically utilized that are required for the OCPW regime, with cell
diameters on the order of B10 mm. Figure 3 explicitly illustrates
these two different conditions, where l - D (Fig. 3a,b) and
lBD (Fig. 3c,d).

Unique to this work is the interaction between attractive
interparticle forces and those confining particles to nodal
positions in a manner that does not arise when l � D. In this
case, the forces of acoustic radiation pressure and interparticle
Bjerknes forces both have the same effect, namely particle
aggregation at nodal locations (see Fig. 3a,b). Conversely, in the
case where lBD, with a single particle per nodal position,
these forces oppose each other. Unique to this study, it then
becomes important to consider the force magnitudes of these
counteracting effects to isolate individual particles. The equations
used to determine the primary acoustic radiation force FR and
the inter-particle Bjerknes force FB for a 2D standing wave are
given by 42

- �

0 Vb l
pp2 fðb; rÞsinð2kxxÞsin 2kyy

0

FRðx; yÞ ¼ - pp2

0 2Vb l
2l

-
2kyy ;

f ¼ 5rp � 2rl - bp

2rp þ rl bl

ð1Þ

;

10 0

10 –1

10 –2

10 –3

10 –4

10 –5

10 –6
10 2 10 3 10 4 10 5

_�_ –1

Ref. [18]
Ref. [19]
Ref. [20]
Ref. [21]
Ref. [22]
Ref. [23]
Ref. [24]
Ref. [25]
Ref. [26]
Ref. [27]
Ref. [28]
Ref. [29]
Ref. [30]
Ref. [33]
Fig. 6
OCPW

Figure 2 | The patterning frequencies in comparison with previous
acoustic trapping work. The bulk of research in acoustic microfluidics
has focussed on the limiting case where the particle diameter is
substantially less than the acoustic wavelength, with D - l and fpl � 1,
with typical frequencies of 1–50 MHz. Here, individual particles can be
separated in individual acoustic force minima through the imposition of
higher ratio of D/l, a regime accessible due to the higher absolute
frequencies utilized here, in the range of 100–230 MHz. The OCPW
regime, which delineates the permissible D/l and absolute values of l,
for trapping of cells (1 mmoDo20 mm) is encompassed by the dotted
line shown.

Thousands
of particles

Single
particle

_P_

_�_ >> _D_ Particle direction

Antinode Node Antinode

_�_ ~ _D_

_t_ = 0
_t_ = _T_ /2
Particle

_x_

Figure 3 | Comparison of the number of particles trapped by large and small acoustic wavelengths. A field with a characteristic wavelength on the order
of the captured cell or particle diameter is required to pattern them individually. Looking at the simplified one-dimensional case, (a) given a relatively large
wavelength (l - D), multiple particles can cohabit in the same nodal position. (c) Although other considerations addressed in the text are important,
individual particles can be spatially isolated given a small-enough acoustic wavelength proportional to the particle diameter (lBD). (b,d) The practical
realization in a 2D field of these different regimes, patterning 10-mm particles in a 2.5-MHz field (reproduced with permission from ref. 26, Copyright 2007,
Acoustical Society of America) and a 100-MHz one (from this work). Scale bar, 300 (b); 36 mm (d). Adapted from ref. 70.

and

6 ~~4~~

vðx; yÞ 2 6rld 4

2� �2

rp - rl ð3cos 2 y � 1Þ
FBðx; yÞ ¼ 4pR 6 6 ~~4~~

- �2

75;

- 2 rl bp - bl

pðx; yÞ 2
9d 2

where R is the particle radius, V is the particle volume, d is the
distance between particles, y is the angle of the particle
orientation relative to the wave propagation, p0 is the pressure
field magnitude, v(x, y) and p(x, y) are the spatially varying
velocity and pressure fields, respectively, and rp, rl, bp and bl are
the densities and compressibilities of the particle and liquid,
respectively. Both interparticle and standing-wave radiation
forces, with FpR 6 and FpR 3, respectively, should be
considered so as to select the range of acoustic wavelengths that
form patterns with particles occupying adjacent nodal locations
in the field, namely the ratio of l/D in which single-particle
patterning is possible. At the upper end of this range (that is,
l44D), more than one particle can inhabit the same half
wavelength. On the other hand, in the case of too small a
wavelength, then particles at nodal positions in the field results in
a FB that is dominant over FR if other particles are in the vicinity.
This would result in particle aggregates, even though the FR field
itself remains periodic. These wavelength limits are explored for
the polystyrene particles and red blood cells (RBCs) used in this
work in Supplementary Fig. 1, where the magnitudes of the
opposing forces |FR|, |FB| are considered and an estimate of the
OCPW-trapping regime is explored. In addition, as periodic
Rayleigh streaming vortices will occur as a result of nonlinear
acoustic effects 43–46, it is important to address their potential
impact on particle trapping. Muller et. al. 47 derived the
expression for critical particle diameter, above which particle

motion is dominated by the primary radiation force and below
which the dominant mechanism is streaming-induced drag; for
polystyrene particles (rp ¼ 1,030 kg m � 3, bp ¼ 3.3 � 10 Pa � 1 )
immersed in water at 100 Hz, this critical diameter is B200 nm,
substantially smaller than those utilized here.

It is important to note that equations 1 and 2 have been
analytically derived in the limiting case where D - l. In the case
here, where DBl/3, the forces predicted by these equations are
expected to deviate somewhat in both their scaling and absolute
magnitude. There does not exist presently, however, analytical
derivations of standing-wave acoustic force in the intermediate
regime explored here, where the particle diameter is of the same
order as the acoustic wavelength; most of the work in the acoustic
microfluidic field has explored cases where D=l � 1 (Fig. 2).
Nor, indeed, is it a trivial task to develop analytical expressions in
this regime, as the force scaling transitions in a nonlinear manner
from FpR 3 to FpR 2 (ref. 48), though it should be possible for
numerical methods with appropriate boundary conditions to
bridge the gap between these two limiting cases. To obtain a
preliminary empirical understanding in this intermediate regime,
the observed particle-trapping regimes are compared with that
expected from the theoretical predictions (Supplementary Fig. 1)
in Fig. 4.

2D particle patterning. A major limitation of many acoustic
patterning methods is that the effective seeding efficiency of the
patterned grid is determined by the initial particle concentration;
with too high a concentration, either multiple particles are
trapped in the same intended space or will be located in between
trapping locations. Too low initial concentrations lead to only
sparse particle patterning. Figure 5 shows the methodology used
to overcome this limitation, one which is made available by the
ability to address individual SAW devices. The acoustic field can
be biased in one direction by applying a reduced SAW amplitude
to one set of transducers, thus creating a partial travelling
wave that pushes suspended particles in the direction of the

_�_ / _D_ = 3.62 _�_ / _D_ = 3.58 _�_ / _D_ = 3.53 _�_ / _D_ = 3.49

_�_ / _D_ = 3.45 _�_ / _D_ = 3.41 _�_ / _D_ = 3.37 _�_ / _D_ = 3.33

_�_ / _D_ = 3.30 _�_ / _D_ = 3.26 _�_ / _D_ = 3.22 _�_ / _D_ = 3.19

Figure 4 | Effect of k/D on particle patterning. As suggested here, there is a limited range of k/D in which single particles can be patterned. The
particle capture efficiency is shown for 6.1-mm polystyrene particles for a number of l/D values, corresponding to odd-number frequencies in the range
163–185 MHz at an applied power of 0.38 W. Particle capture and patterning is limited outside of the range 3.2Zl/DZ3.6. Fluorescent particle images
(250 � 250 px) are obtained from the same central chamber region. Scale bar, 30 mm.

a b c

PDMS

H2O

Figure 5 | Methodology to increase seeding efficiency. (a) A solution of 6.1-mm particles suspended in water is (b) exposed to a l ¼ 21-mm,
0.52-W-applied power, 171-MHz two-dimensional acoustic field biased in one direction. This biased field translates particles across the array through the
force of the partial travelling wave thus created, effectively ‘bumping’ particles along so that only one particle occupies a given nodal position (see inset),
pushing most excess particles to the chamber wall in the direction of bias. (c) The excess particles can then be removed from the chamber by pumping fluid
into the chamber, manipulated either by hand or a syringe pump. The majority of patterned single particles, however, remain trapped. Scale bar, 100 mm.

higher-amplitude wave propagation, while the 2D force potential
minima ‘sieve’ out individual particles. Here, the travelling wave
force acts locally on particles against the partial standing-wave
force, which is of sufficient magnitude and appropriate dimensions to retain individual particles but not groups of them,
resulting in a seeded particle array with excess particles to the
chamber wall. These latter particles can subsequently be removed
with the application of a fluid flow, while single particles remain
trapped in the acoustic array.

As discussed in the previous section, the acoustic wavelength
used to pattern particles is important to operate in a regime
where only one particle can inhabit a given space due to
steric considerations, but particles are not so close that
inter-particle forces dominate over that arising from standing
waves. To characterize this system, polystyrene particles with
D ¼ 5.1–10.0 mm are introduced into SAW devices whose
wavelengths correspond to their pattering in Fig. 6a, where
particles are patterned in the minimum-force potential (nodal)
locations of the acoustic force field. A scan of the surface
displacements on the SAW substrate is shown in Fig. 6b, where

the time-averaged nodal (minimum displacement) and antinodal
(maximum displacement) positions can be seen. The alignment of
particles at the nodal positions in the horizontal plane has been
explored previously by Collins et al. 49 . Figure 6c confirms the
relationship between the observed inter-particle wavelength and
the applied acoustic one, namely that lSAW ¼ lp, the SAW and
particle wavelength, respectively. Further, a linear relationship
exists between the range of wavelengths that can be used to trap
individual particles in square patterns and the dimensions of
particles in those patterns; Fig. 6d shows the optimal trapping
wavelength lopt for a range of particle diameters, showing
loptpD. Interestingly, particles and cells will self-segregate from
cluster formations if the frequency is ramped up or down from to
the optimal trapping wavelength; Supplementary Movie 1 shows
the automatic grid arrangement and separation of lymphocyte
cells (to be discussed in detail in the following section), as the
frequency is continuously increased from 110 MHz (l/DE4) to
126 MHz (l/DE3.5).
The shaded blue region of Fig. 6d corresponds to the range of
wavelengths that are empirically determined for polystyrene

a 5.1 μm 6.1 μm 6.6 μm b

SAW

5.1 μm

6.1 μm 6.6 μm

7.0 μm 7.2 μm 10.0 μm

_�_

Antinodes

Nodes

_�_

c d

1.15

1.1

1.05

0.95

0.9

0.85

_�_ p= _�_ SAW _F_ R> _F_ B

Fig. 4
Data 40 Fig. 6a

_�_ p= _�_ SAW

0.8 15
5 6 7 8 9 10 11 5 6 7 8 9 10 11

_D_ (μm) _D_ (μm)

Figure 6 | Characterization of the OCPW-patterning phenomena. (a) Spherical particles of essentially any size can be patterned in the two-dimensional
acoustic grid, here showing the patterning of 5.1-, 6.1-, 7.0-, 7.2- and 10-mm polystyrene particles in acoustic fields of different wavelengths actuated by a
204-, 171-, 149-, 145- and 101-MHz (respectively) signal at 0.38 W. Scale bar, 30 mm. (b) The position of these particles is dictated by the nodal positions in
the acoustic field, with particles migrating to the minimum displacement locations in the field. The image shown here is a map of the maximum surface
displacement on a l ¼ 30-mm device as measured by a laser doppler vibrometer. Scale bar, 15 mm. (c) As expected, the distance between the patterned
particles in a is proportional to the wavelength of the acoustic field, with lp/lSAW ¼ 1, where lp is 2 � the distance between particles in the x- or
y-directions. (d) The OCPW-trapping region (as measured by l/D) scales linearly with particle diameter, although the range of these values is somewhat
smaller than predicted by equations 1 and 2 (see Supplementary Fig. 1), suggesting that these equations may lose some predictive power as the particle
dimensions approach that of the acoustic wavelength. Error bars in c and d denote one s.d. of the measured particle spacings in a. Adapted from ref. 70.

particle patterning, where Fig. 4 shows the effect that changing
l/D has on the seeding efficiency of the particle grid. Here, this
range 3.2Zl/DZ3.6 is somewhat smaller than the 3.0Zl/DZ
4.0 range suggested from the theoretical prediction where FR4FB
(Supplementary Fig. 1a), which corresponds to the shaded grey
region in Fig. 6d. This is not entirely unexpected and suggests that
the standing-wave force may drop off more quickly than the
inter-particle Bjerknes force as the particle diameter approaches
that of the wavelength. Indeed, this stands to reason whether one
considers that the force acting on a particle is the sum total of all
the force contributions from a spatially varying acoustic field; the
maximum acoustic force occurs at the region of the highest force
gradient. Because a particle size above the simplifying case where
D - l will access a wider range of force gradients, all of which
will be less than that at the nodal position, the net total of those
forces will be less than that if only the force at the nodal position
is considered. This concept, however, requires further exploration
looking specifically at the magnitude of force generation for
different l/D.

The maximum theoretical width of the chamber in which
particles can be patterned is ultimately determined by the relative
magnitude of the local standing-wave and travelling-wave forces,
and is discussed in Supplementary Note 1.

Cell patterning. Patterning with cells presents challenges
and opportunities when compared with rigid synthetic particles.

On one hand, the deformability of cells (especially those without
cell walls) allows them to co-locate in acoustic wells for l/D that
would not be available to polystyrene spheres. However, as can be
inferred from equation 2, their greater compressibility means that
cells can typically be brought much closer together before
Bjerknes forces dominate and bring them into direct contact.
When suspended in isotonic conditions the RBC diameter is
B7.5 ± 0.5 mm (ref. 50), although with a thickness of only about
2 mm it is potentially more difficult to pattern these cells without
instances of co-localization. Moreover, to better compare directly
with spherical polystyrene particles and cell types that assume a
mostly spherical shape in suspension, RBCs are suspended here in
0.8 � PBS, to inflate them to an oblate spheroid shape (diameter
approximately 6–7 mm) as opposed to their normal biconcave
disk shape, a transformation that has been explored previously 51 .
Figure 7 shows the effective capture and patterning of individual
RBCs over a range of l/D wider than that for the polystyrene
spheres in Fig. 6, with 2.5Zl/DZ4.0. This is important for
capturing large numbers of individual cells, given the natural
variability in cell dimensions between cells at the same or
different stages in development.

Even though RBCs are non-uniform in shape, unlike the case
for most cells in suspension 52, it is still possible to pattern nonspherical cells, in this case to produce 2D RBC cell patterns in
their biconcave disk shape, despite the substantially smaller
acoustic force that can be imparted (as FR is proportional to the
cell volume). To illustrate the potential of the OCPW system for

a b c

_�_ / _D_ ≈ 2.5 _�_ / _D_ ≈ 3 _�_ / _D_ ≈ 4

Figure 7 | Red blood cell patterning. One cell per acoustic well (OCPW) can be produced for a wide range of wavelengths with respect to cell
diameters l/D. Here, red blood cells made semi-spherical by suspension in an hypotonic solution (0.8 � PBS) and patterned in a (from left to right);
(a) 15 mm, (b) 18 mm and (c) 25 mm field (232, 200 and 144 MHz, respectively, at E0.5 W). While geometric considerations determine the lower limit of
l/D, where two cells cannot occupy the same space, the ability to pattern OCPW becomes strongly determined by the initial location of cells at the upper
limit, theoretically at l/D ¼ 4, above which multiple cells can co-locate in the same position. Scale bar, 50 mm.

Figure 8 | 2D patterning allows for the spatial isolation and identification
of individual cells. Here, a green fluorescent protein-expressing malarial
parasite Plasmodium falciparum has infected a small proportion of a
population of red blood cells (RBCs). In patterning this population in
an 18-mm acoustic field (201 MHz at E0.5 W), infected RBCs can be
independently visualized. Scale bar, 40 mm.

studying low-frequency events, Fig. 8 shows captured RBCs, a
fraction of which are parasitized by Plasmodium falciparum, here
in the trophozoite phase. A strain of the parasite expressing green
fluorescent protein is used to visualize and identify independent
infected cells. Here, a 4.5-mm-high chamber is used to constrain
cell movement in the vertical direction, in addition to the acoustic
force field used in the horizontal plane; as opposed to the case
with spherical RBCs patterned in a 10-mm chamber as in Fig. 7,
highly deformable and thinner normal RBCs are more likely to
co-locate in the same horizontal position. More broadly, this
raises the point that the chamber height should be approximately
on the order of the minimum cell dimensions, to avoid cells
occupying multiple nodes in the vertical direction; this is less of a
concern for polystyrene particles, however, where stronger
scattering results in stronger particle–wall interactions that
preferably favours certain trapping locations in the case of a
non-symmetrical field in the vertical direction (with the
piezoelectric substrate (LN) and polydimethylsiloxane (PDMS)
as the chamber floor and ceiling). A consequence of using planarshaped, highly deformable RBCs can be seen in some locations in
Fig. 8, where a number of cells contact edge-to-edge despite being
trapped. Here, it is hypothesized that these cells can reconfigure
in response to the local pressure conditions, allowing small
sections of cells to contact adjoining cells; this behaviour is more
likely to occur in localized areas of the chamber, in which the
SAW field is stronger in one direction due to non-uniformity

in amplitude across the width of the IDTs (as noted in ref. 53).
The larger diameter of flat RBCs may also contribute to the
observed behaviour, where sphericalized RBCs will have a
marginally smaller diameter (measured as E6.5 mm versus
7.5 ± 0.5 mm for the literature value of flat RBCs 50 ).
Any cell-trapping technique has the potential to impact cell
viability, through induced shear strain or local heating, for
example. The biocompatibility of MHz-order acoustic fields,
important for long-term cell studies, has been the subject of recent
study 23,32,54–56, where viable cells have been trapped in nodal
positions for durations on the scale of minutes to hours 18,22,57,58 .
These studies are further validated in Fig. 9, which shows the
patterning of human peripheral blood mononuclear cell
lymphocytes tagged with carboxyfluorescein succinimidyl ester,
a long-life cell membrane leakage assay used to track viability and
movement of these cells (Fig. 9a inset). While acoustic power
levels in excess of that required for stable trapping are sufficient to
rapidly lyse these cells (Fig. 9b), cell lifespan rapidly improves for
lower powers, establishing (for our system) an effective threshold
of B570 mW, above which most lymphocytes will quickly lyse
and below which cells can maintained in the acoustic field for
long periods. Figure 9c shows that viability can be maintained
over a period of hours, with B80% of cells alive after 2 h at
320 mW, and with negligible difference to the control sample at
220 mW, a power level shown in Fig. 9a and Supplementary
Movie 2 to be perfectly capable of capturing the cells. These
results are indicative of a typical human cell lymphocyte; however,
different cell types will have different lysis threshold powers;
individually patterned RBCs, which do not contain cell nuclei, are
able to retain their morphologies over similarly long periods at
powers of 500 mW (the power used in Figs 7 and 8).

Batch processing of cells is made possible as the acoustic field
can be arbitrarily applied. This temporal control of the force field
allows cells to be patterned, analysed and released. A short
demonstration of this, where suspended RBCs are patterned and
then released on-demand in a continuous flow is shown in
Fig. 10a and Supplementary Movie 3. Figure 10b shows that this
ability to hold particles and cells in a continuous flow can be
extended to exchange of medium, either to clear metabolites or
introduce reagents so cellular response can be assayed. Array
filling is also possible in a continuous flow, as Supplementary
Movie 2 demonstrates, here with individual human lymphocytes
automatically captured in nodal locations.

The OCPW system can also be used for other activities that
would be difficult or impractical to realize in the mechanical
trapping platforms previously demonstrated 59,60, where paired
cells are of similar dimensions. Figure 10c explores the limits
of co-locating particles with significantly different sizes, where
10-mm particles are patterned in the conventional 2D grid and

a b

c d

1.5

0.5

0.8

0.6

Cell death

0.40 2 4 6 8
_t_ (s)

10 μm + 6μm 10 μm + 5μm 10 μm + 4 μm

10 μm + 3 μm 10 μm + 2 μm 10 μm + 0.5 μm

0
500 1,000 1,500 2,000

_P_ (mW)

100

220 mW
320 mW

40
0 2 4 6 8

_t_ (h)

Figure 9 | Lymphocyte trapping and viability. (a) Human peripheral blood
mononuclear cell (PBMC) lymphocytes are simple to pattern as compared
with RBCs due to their spherical shape. Inset shows a fluorescence image of
carboxyfluorescein succinimidyl ester (CFSE)-infiltrated lymphocytes, a
membrane leakage assay that demonstrates cell viability. Cells are
patterned in a 126-MHz field from a continuous flow at 0.5 ml min � 1 at
0.22 W, from an input concentration of 4,500 cells per ml to a local
patterned concentration B100 � this. Supplementary Movie 2 shows
individual cell capture in the array. Scale bar, 50 mm. (b) The power level
limits the maximum exposure time, with the inset showing an example
control-normalized fluorescence intensity measurement of membrane
integrity loss (field applied at t ¼ 0). The time to lysis is strongly dependent
on the applied power: although all continuously monitored cells (population
(pop.) 11) are lysed within 49 s at 890 mW, only one is observed to do so at
570 mW within the E5-min period before photobleaching rendered the
sample unusable, as denoted by (*). Error bars denote 1 s.d. for observed
death times of 11 cells at 890/1,280/1,740 mW. (c) Below this threshold
power (B1 mW mm � 2 ), cells can be trapped for increased periods with
negligible effects on cell viability, here showing the long-term comparison
of viable cells (pop. 50/48 cells for 220/320 mW studies) to a control
(cells from same population incubated simultaneously in identical chamber
without SAW, total pop. 57). Photobleaching of the sample (at *) is avoided
for this duration through selective shuttering of the light source.

Figure 10 | On-demand patterning with media exchange and particle colocation. (a) RBCs in a continuous flow can be (b) individually patterned
and held for an arbitrary length of time through the imposition of an
externally applied acoustic field (229 MHz at 0.25 W). The ability to hold
particles and cells in place can further be used for applications such as
media exchange, where c and d show the same trapped 6.1-mm particle grid
suspended in water with a dye solution injected from a separate inlet,
although some reconfiguring occurs due to changing local flow conditions
(171 MHz at 0.52 W). Arrows between panels (a/b) and (c/d) denote
interchangeability between random/patterned cells and suspension in
different media, respectively. Using a patterned array of 10-mm particles,
smaller particles can be trapped and co-located in the same locations,
useful for cell-pair interaction studies where there is a large size difference
between cells. (e) The limits to this activity, as defined by the difference
between the size of the larger and smaller particle. When the smaller
particle is larger than E50% of the larger particle’s size, particles can either
bridge between trapping locations (double-lined arrow) or cluster in a nodal
location independently (solid arrow). Below E30%, particles can access
trapping locations near the chamber roof offset from the primary trapping
locations (dashed arrows). (a–e) Scale bars, 50 mm.

where smaller 3–5 mm particles (B30–50% of the larger
particle size) are preferentially retained and co-located in the
same positions due to both inter-particle and primary radiation
forces. Outside of this range, particles will either preferentially
form clusters or trap in secondary locations offset to the primary
field near the chamber extremes. Although these secondary
locations are not the focus of this work, it is thought that the

differential effect acoustic streaming has on smaller particles
plays a role in this separation, as explored elsewhere 41,61 . This
ability has the potential to be used in the study of single-pair
bacterial/eukaryotic cell interactions, as a single-cell pairing
extension of en masse cell culture work in the field 62 .

Finally, the range of patternable l/D values gives an indication
of the required monodispersity of patterned population. For
polystyrene particles, the range of 3.2Zl/DZ3.6 corresponds to a
range of particle diameters ± 6% relative to the mean Davg that
are permissible; the particles used here meet this criteria, which
have a manufacturer-supplied coefficient of variation E3%. Cells,
for which similar trapping results can be obtained over a wider
range of l/D, can accommodate a similarly wider range of
diameters; for the RBCs tested, the 2.5Zl/DZ4.0 range
corresponds to BDavg ± 20%, which allows even moderately
polydisperse populations to be trapped with OCPW.

Discussion
In summary, we demonstrate a technique for the active pattering
of spatially isolated individual cells in an acoustic field defined in
two dimensions, including a characterization of the regime where
OCPW patterning is possible. This is realized through the
previously unexplored imposition of an acoustic field whose
wavelength is of the same order of the cell dimensions, where
only one particle can inhabit a given nodal location due to steric
constraints. Rayleigh-type SAW wavemodes are uniquely suited
to operate in this regime, where high-frequency fields are used to
generate wavelengths on the scale of microns. Moreover, SAWdriven OCPW patterning is a biocompatible technique for the
observation of single cells over long time periods that, in contrast
to hydrodynamic/mechanical cell-pattering methods, is capable of
re-use and batch processing. This latter capability is especially
useful for the analysis of rare cells that may only make up a
fraction of the total cell population. By patterning cells in spatially
distinct positions, individual cells can be optically analysed over
long periods in isolation from each other, a difficult task in
conventional cell culture. Through the process of spatially
isolating individual cells and constraining their positions, the
activity of individual cells can be assayed, as opposed to the case
in conventional bulk cell culture where parameters are effectively
averaged across an entire population. This is especially
important in pathogen biology, where infectious agents such as
Mycobacterium tuberculosis can express many different phenotypes in a given population, relevant to antibiotic resistance 63 .

The ratio of wavelengths to diameters is crucial to single-cell
patterning. In contrast to previous work where the acoustic
wavelength is an order of magnitude or more greater than the
particle or cell dimensions, reducing the acoustic wavelength
sufficiently allows the patterning of individual, rather than
clumps of particles. It is found that this single-particle-patterning
effect is only workable for a relatively narrowly defined band of
l/D ratios, however, due to the competing influence of the force
generated from the acoustic standing-wave field and those
resulting from inter-particle interactions. Encouragingly though,
cells are far less sensitive to inter-nodal spacing than synthetic
particles, where OCPW can be achieved for a substantially larger
range of wavelengths. Here, individual RBCs are spaced between
7.5 and 12.5 mm from each other, opening the door for future cell
studies where inter-cellular distance is an important parameter.
In addition, as batch processing is possible through the tunable
application of the acoustic field, hundreds or thousands of cells
can be analysed in a short period of time in a repeated fashion.
This makes this method suitable for the isolation and analysis
of phenomena affecting a small fraction of a cell population.
Although outside the scope of this particular work, future

applications for the OCPW-patterning system include long-term
capture and observation for drug discovery, where short- and
long-term cellular response to effector molecules can be assessed.

Methods
Device fabrication and set-up. In this work, four sets of IDTs, consisting of a
200-nm-thick Al conductive layer deposited on a 7-nm-thick Cr adhesive layer,
are patterned on a piezoelectric 128� Y-cut lithium niobate (LN, LiNbO3) substrate.
LN is often used in SAW microfluidic applications due to its high coupling
coefficient and resultantly large surface displacements. Further, 300 nm of SiO2
is deposited on the substrate to prevent corrosion of the IDTs and to enhance
channel bonding. Four sets of transducers, each rotated 90� around the focal region
from neighbouring sets, are used to generate a two-dimensional sound field in
which particles are patterned. Importantly, each transducer’s SAW propagation
axis is offset 45� relative to the usually preferred x-propagation direction. This
orientation is chosen because the coupling coefficient is equivalent in all directions
symmetric about this axis of the crystal, so that an equivalent number of finger
pairs are required to produce an equivalent amplitude wave for each of the four sets
of IDTs, although the sound speed in this direction is marginally lower at this
offset, with csE3,590 m s � 1 instead of csE3,960 m s � 1 in the x-direction 64 .
A 480 � 480-mm polydimethylsiloxane chamber (280 � 280 mm in Supplementary
Movie 2) is created using soft lithography from an etched silicon mould and
bonded to the LiNbO3 device using plasma-activated surface treatment. This
chamber width is smaller than the 750 mm IDT aperture to maximize the
uniformity of the acoustic magnitude within the chamber. In addition, to maximize
the acoustic energy transferred to the liquid in the chamber, rather than to the
PDMS that comprises the chamber structure, the IDTs are completely enclosed in
an air-filled chamber with only a 56-mm-wide PDMS partition separating the
respective air and liquid sections. The device is then actuated in all four channels
by a signal generator (Belektronig powerSAW f20 for particle experiments, Hameg
8134–3 with Amplifier Research 25A250A for cell experiments), with a biased field
produced by selective attenuation (with a 5-kO variable resistor) with particle and
cell patterns viewed through a fluorescent microscope (Olympus BX53).
Fluorescent polystyrene particles, all with coefficient of variation E3%, are
obtained from Magsphere, USA, and RBCs are obtained from an anonymous
healthy donor. To help prevent particle adhesion to the chamber walls, polystyrene
particles are suspended in a 0.2% solution of polyethylene glycol. Surface
displacement measurements are made by applying an a.c. signal at 0.126 W
(Rohde&Schwarz SMBV100A) and measuring the resultant surface velocities with
a laser doppler vibrometer (Polytec UHF-120).

P. falciparum parasite culture. RBCs are obtained from the Australian Red Cross
and suspended in 1 � PBS. Hypertonic solution is made with 4:1 1 � PBS/1%
polyethylene glycol solution to make the RBCs spheroid. The P. falciparum parasite
strain used in this study is D10/ACP signal-green fluorescent protein tagged 65 and
cultured in vitro 66 . For analysis, the cultures are synchronised to ring stages using
5% sorbitol 67 and collected 24 h later. Temperatures of 35 ± 2 �C are maintained on
the microfluidic chip via a thermoelectric device on which the OCPW system is
mounted, where steady-state temperatures are typically achieved on the order of
seconds 68,69 .

Peripheral blood mononuclear cells. Peripheral blood mononuclear cells
(PBMCs) are isolated from blood collected in EDTA vacutainer tubes (BD
Biosciences, USA) from healthy volunteers at the Monash Precinct at the Alfred
Hospital, Melbourne, Australia (Human Ethics Committee Approval 2007002182)
via Ficoll density centrifugation (GE Healthcare, Sweden), and washed three times
in Dulbecco’s phosphate buffered saline (dPBS, Sigma-Aldrich, USA). PBMCs are
then fluorescently labelled by incubation for 5 min at in 0.4 mM carboxyfluorescein
succinimidyl ester in dPBS at 37 �C at 10 6 cells per ml, followed by quenching in
ice-cold PBS media supplemented with 10% FCS (P10). Cells are then washed
at 4 �C by centrifugation with P10, and resuspended to a final concentration
of 150 ml/10 6 cells in P10 for staining for 15 min with an APC-Cy7-labelled
monoclonal antibody against CD4 (anti-CD4)(BD Biosciences). After further
washing, cells are resuspended in P10 and cell sorted using flow cytometry
using forward scatter versus side scatter gating for resting lymphocytes followed by
selection of double-stained APC-Cy7 anti-CD4 (650/785 nm excitation/emission)
and carboxyfluorescein succinimidyl ester (488/525 nm excitation/emission)positive cells on the FACSAria high-throughput platform. The flow-sorted cells are
washed and resuspended at 4.5 � 10 6 ml � 1 in cold P10, and kept in at 4 �C until
application to the SAW device.

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Acknowledgements
This work is partially supported by the Monash Interdisciplinary Research Grant. Principle
fabrication of the OCPW devices was performed at the Melbourne Center for
Nanofabrication, the Victorian node of the Australian National Fabrication Facility. The
GFP-tagged P. falciparum line was kindly supplied by G. McFadden. Special thanks to
Samuel Martin for his SolidWorks modelling assistance, and Mutsa Madondo for help
with CSFE loading and flow cytometry cell sorting.

Author contributions
D.J.C. is the main contributor who planned and initiated the experiments, fabricated
devices, collected and analysed data and prepared the bulk of the manuscript.
A.N. is the coordinator and principle investigator, B.M. performed cell culture and
preparation procedures and J.G.-B., C.D. and M.P. provided expertise in cell culture

and handling. B.M. and M.P. wrote sub-sections for P. falciparum and PBMC culture/
preparation, respectively, and all parties contributed to the review and editing of the
manuscript.

Additional information
Supplementary Information accompanies this paper at [http://www.nature.com/](http://www.nature.com/naturecommunications)
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Competing financial interests: The authors declare no competing financial interests.

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How to cite this article: Collins, D. J. et al. Two-dimensional single-cell patterning
with one cell per well driven by surface acoustic waves. Nat. Commun. 6:8686
doi: 10.1038/ncomms9686 (2015).

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