The use of ultrasound to manipulate bioparticles has already been shown to work in principle and to be safe. This field is now rapidly developing, but existing systems are limited by their reliance on resonance between piezoelectric plates, in turn fixing the nodal points towards which particles move, or their need to first trap particles then physically move the transducers. This article describes an innovative system based on small, easily electronically integrated, ultrasonic transducers.
by Dr Dave Hughes
Introduction
Modern day life scientists have many tools at their disposal for probing the inner secrets of cellular life. Despite numerous recent advances in molecular biology and microscopy, which allow investigation of dynamic processes in cells, including manipulation of molecules using optical tweezers, there is still a need for a quantitative device that can accurately manipulate and characterise forces and mechanical properties of single cells and tissues.
There has already been much work carried out that has shown that acoustic waves can be used to levitate masses of up to a kilogram, and while this is certainly too heavyhanded for working with 10-50 micron diameter cells, research is underway to investigate the use of similar techniques to produce a device for the modern biology lab.
Forces from sound
An acoustic wave is a series of pressure oscillations travelling through a medium, with sound being an oscillation within the frequency range that humans can hear. Ultrasound is defined as any acoustic wave with a frequency above 20 kHz. When an ultrasound wave propagates through a medium, the pressure oscillates with time and position and by introducing boundaries between different media, a standing wave can be created that confines the pressure oscillations in space.
If a material had only linear properties i.e. had identical compression and expansion rates, then these pressure oscillations would average out to zero. However, most materials have some degree of non-linearity, which gives rise to a non-zero time-averaged pressure that produces forces which can be exerted on particles, as shown in Figure 1.
These forces, as described by Gor’kov [1], are a function of the relative acoustic properties of the particle and the medium surrounding it. Known as primary acoustic forces, they tend to push particles of higher density towards areas of low pressure. There is a secondary acoustic force which acts perpendicularly, and to a lower degree, to the primary force [2]. It arises out of variations in the medium and inter-particle interactions. The dominant primary acoustic force has been shown to increase with the frequency of the ultrasound source and particle size, with greater volumes producing greater force.
Ultrasonic forces are typically greater than those which can be produced by optical and dielectrophoresis traps, with magnitudes of nanonewtons predicted as possible [3]. The dependence of the ultrasonic force on particle dimensions also improves on optical traps, as it is possible to move objects up to 50 μm in diameter.
Current applications
Ultrasonic standing waves have a long history of being used to manipulate particles and cells; as a result, a full lite rature review is beyond the scope of this article.
Recently, researchers at Pennsylvania State University, USA have demonstrated a surface acoustic wave device that can position blood cells in line and grid patterns [4]. This device utilises a pair of inter-digital ultrasound transducers on a piezoelectric surface to produce a standing surface acoustic wave across a fluid chamber. Cells entering into the chamber with microfluidics are displaced towards the pressure nodes. This is achieved with the same ability as optical traps, but using a fraction of the energy. Manipulation like this could in the future be coupled to a functional analysis of some of the cells' properties
The placement of cells in polymer gels is a method of carrying out microbial analysis, as it allows cells to be immobilised while under investigation. Ultrasonic standing waves have been demonstrated as a method of patterning the cells inside polyacrylamide and agar gels before the gels set [5]. For this work, a cylindrical ultrasound transducer is placed around a tube containing the un-set gel and the cells. This transducer produces a series of concentric nodal pressure rings through the gel, towards which the cells are forced. The use of ultrasonic standing waves has been shown to have no ill effects on the viability of the cells because the cells are placed in the areas of low or no pressure [6].
In a similar geometry, a cylindrical ultrasound transducer has been added to a commercial flow cytometry system to increase sensitivity. The Attune Acoustic Focusing Cytometer uses the transducer to produce a thin stream of cells for optical measurement. This increases sensitivity because the increased positional accuracy with which the cells pass through the light source leads to a reduction of noise in the measurement, with no reduction in throughput velocity.
Researchers at the University of Southampton, UK have developed a device, shown in Figure 2, which produces a one-dimensional pressure gradient that decreases towards a cover-slip which seals a fluid chamber [7]. The resulting gradient force is orientated towards a point inside the cover slip. The device is able to sort cells by size using parametric flow through the chamber, with larger particles experiencing a greater deflection than smaller particles. The device also has microfluidic sensing applications if molecules are pressed against an assay surface for chemical investigation.
New frontiers for ultrasonic manipulation
Previous research has focussed mainly on static fields which rely on resonance arising in the geometry of the manipulation systems. A new collaboration between the British Universities of Dundee, Glasgow, Bristol and Southampton, along with several partner companies, aims to create a new class of ultrasonic device called an “Electronic Sonotweezer”. These devices are being designed to allow the position of pressure nodes to be controlled electronically through multi-transducer or ultrasonic array systems, removing the need to manually move the ultrasound source, as is the case with current systems. Figure 3 shows a series of images demonstrating the concept, with a particle first levitated then moved laterally using a multi-transducer setup.
The Sonotweezers consortium is currently testing a prototype device that makes use of a 30-element micro-array, which has been fabricated using industry standard techniques allowing for scalability. The diagram shown in Figure 4 outlines a reconfigurable ultrasonic sorting device that would be possible with such an array-based device. This type of device has obvious parallels with advanced optical tweezers that make use of spatial light modulators to create multiple trap geometries. It will allow very fine control of manipulation, involving no moving parts, scaling down to micro-level, but maintaining the higher forces available from ultrasound.
Researchers at the University of Dundee are investigating the use of such a device to measure the motility forces of cells directly in a method similar to that used with optical traps on smaller molecules. Work previously carried out on the social amoeba Dictyostelium discoideum has shown that the forces that a single cell can generate during motility are of the region of nanonewtons (x10-9N). This is above the limit of optical traps and therefore provides the motivation to use ultrasonic fields in which the forces produced should be much greater.
Another application that is being investigated is the use of acoustic fields for the measurement of rheological properties of cells. A one-dimensional standing wave device, as shown in Figure 2, is being used by the researchers in Dundee to probe the properties of the cell cytoskeleton by deforming and relaxing cells with the ultrasonic force.
Conclusions
Ultrasonic manipulation has been established as a viable phenomenon for many years, but has yet to gain widespread use in the biology lab.
The advent of smaller, more advanced, multi-element ultrasound sources provides promise for single cell manipulation and characterisation.
References
1. Gor’kov LP. On the Forces Acting on a Small Particle in an Acoustical Field in an Ideal Fluid. adsabs.harvard.edu/abs/1962SPhD....6..773G
2. Laurell T et al. Chem Soc Rev 2007;36(3):
492-506.
3. Lee J et al. Appl Phys Lett 2009; 95(7):073701.
4. Shi J et al. Lab Chip 2009;9(20):2890-2895.
5. Gherardini L et al. Ultrasound Med Biol 2005; 31(2):261-272.
6. Radel S et al. Ultrasonics 2000;38(1-8):
633-637.
7. Glynne-Jones P et al. J Acoust Soc Am 2009;126(3):EL75-EL79.
Acknowledgements
The author wishes to thank Prof. Kees Weijer at the Division of Cell and Developmental Biology (University of Dundee) for support, and also acknowledges the advice given by Prof. Bruce Drinkwater (University of Bristol) and Sandy Cochran (University of Dundee) when writing this article. The author is funded by the EPSRC.
The author
Dave Hughes, Ph.D
Divison of Cell and
Developmental Biology
College of Life Sciences
University of Dundee
Dundee,
DD1 5EH,
UK
d.a.hughes@dundee.ac.uk
