Three-Dimensional Mid-Air Acoustic Manipulation by Ultrasonic Phased Arrays

The essence of levitation technology is the countervailing of gravity. It is known that an ultrasound standing wave is capable of suspending small particles at its sound pressure nodes. The acoustic axis of the ultrasound beam in conventional studies was parallel to the gravitational force, and the levitated objects were manipulated along the fixed axis (i.e. one-dimensionally) by controlling the phases or frequencies of bolted Langevin-type transducers. In the present study, we considered extended acoustic manipulation whereby millimetre-sized particles were levitated and moved three-dimensionally by localised ultrasonic standing waves, which were generated by ultrasonic phased arrays. Our manipulation system has two original features. One is the direction of the ultrasound beam, which is arbitrary because the force acting toward its centre is also utilised. The other is the manipulation principle by which a localised standing wave is generated at an arbitrary position and moved three-dimensionally by opposed and ultrasonic phased arrays. We experimentally confirmed that expanded-polystyrene particles of 0.6 mm, 1 mm, and 2 mm in diameter could be manipulated by our proposed method.     

One-dimensional acoustic standing waves in rectangular channels for flow cytometry

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry.     

A study of the spatial organisation of microbial cells in a gel matrix subjected to treatment with ultrasound standing waves

Retention and manipulation of microbial cells through exploitation of ultrasonic forces has been reported as a novel cell immobilisation technique. The spatial ordering of yeast cells, within suspensions subjected to an ultrasonic standing wave field, was analysed for the first time. A technique, based on `freezing' the spatial arrangement using polymer gelation was developed. The resultant gel was then sectioned and examined using microscopic techniques. Light Microscopy confirmed the presence of specific regions in the ultrasonic field, where the cells are organised into bands corresponding to the standing waves' pressure nodal planes. Computer Image Analysis measurement of several physical parameters associated with this cell distribution matched the values derived from the theoretical model. The spatial cell-cell re-arrangement within each band and uneven distribution along the nodal planes have been analysed by Scanning Electron Microscopy. These results complement the ongoing study of the process of immobilisation of microbial cells by ultrasound standing waves. This revised version was published online in July 2006 with corrections to the Cover Date.     

Toward mechanical manipulations of cell membranes and membrane proteins using an atomic force microscope

Recent advances in the use of the atomic force microscope (AFM) for manipulating cell membranes and membrane proteins are reviewed. Early pioneering work on measurements of the magnitude of the force required to create indentations with defined depth on their surfaces and to separate interacting pairs of avidin-biotin, antigen-antibody, and complementary DNA pairs formed the basis of this field. The method has subsequently been applied to map the presence of cell surface receptors and polysaccharides on live cell membranes by force measurement, with promising results. Attempts to extract phospholipids and proteins from lipid bilayers and live cell surfaces have been reported, providing a new tool for the manipulation of cellular activities and biochemical analysis at the single-cell level. An increasing awareness of the effect of the pulling speed (nm/s or microm/s), or more accurately, the force loading rate (pN/s or nN/s) on the magnitude of the rupture force, has led researchers to construct energy diagrams of rupture events based on the parameters available from such studies. Information on such nature of the interplay of force and loading rate is vital for nanomanipulation of living cells and cell membranes. Some relevant work for membrane manipulation using other methods is also reviewed in relation to AFM-based methodology.     

Propagation of shear waves in muscle tissue

A mathematical model of the propagation of acoustic shear waves in muscle tissue is considered. Muscle is modeled as an incompressible transversely isotropic viscoelastic continuum with quasi-one-dimensional active tension. There are two types of shear waves in an infinite medium. Waves of the second type (transverse) propagate without decay even when myofibril viscosity is taken into account. A problem of standing transverse waves in a rectangular layer was investigated numerically. The values of the problem parameters are found for which one can easily estimate the active tension (or muscle tone) from the characteristics of standing waves. This value is informative for diagnostics of the muscle state.     

Propagation of shear waves in the muscle tissue

A mathematical model of the propagation of acoustic shear waves in muscle tissue is considered. The muscle is modelled by an incompressible transversely isotropic viscoelastic continuum with quasi-one-dimensional active tension. Two types of shear waves in an infinite medium have been established. The waves of the second type (transverse) propagate without attenuation even when myofibril viscosity is taken into account. A problem of standing transverse waves in a rectangular layer has been investigated numerically. The values of the problem parameters have been found for which the active tension or muscle tonus is easily estimated from the characteristics of standing waves. This value is informative for the diagnosis of muscle state.     

Propagated disturbances of transverse potential gradient in intracellular fibrils as the source of motive forces for longitudinal transport in cells

Summary It is proposed as a working hypothesis that conformational changes propagated like waves along intracellular fibrils (tubules, microtubules, microfilaments) have an electric component,i.e., there are waves of disturbance of electric potential in the fibrils. The paper considers the unavoidable consequences of the wave. The latter is accompanied by local electric field in the boundary layer of cytoplasmic fluid. Both positively and negatively charged particles may be attracted to the fibril in certain regions of the field and, being attracted, the particle may be under the action of longitudinal component of electric force. When the force is strong enough to move the particle with wave velocity, the particle will travel smoothly along the fibril, otherwise the movement will be saltatory or of agitation type. Net electroosmotic flow in one direction in the boundary layer of fluid may be expected when the waves are propagated in series. Turbulent motion of the fluid caused by the waves may provide the basis for activated diffusion. Asymmetry of the wave may account for polar transport of this sort. The electric field transmitted along the fibril across a sieve pore in phloem may facilitate electroosmotically the flow through the pore. Quantitative requirements of the hypothesis that electric field generated by the waves may account for different aspects of longitudinal transport in cells are apparently met.     

A review of the harvesting of micro-algae for biofuel production

Many researchers consider efficient harvesting is the major challenge of commercialising micro-algal biofuel. Although micro-algal biomass can be ‘energy rich’, the growth of algae in dilute suspension at around 0.02–0.05 % dry solids poses considerable challenges in achieving a viable energy balance in micro-algal biofuel process operations. Additional challenges of micro-algae harvesting come from the small size of micro-algal cells, the similarity of density of the algal cells to the growth medium, the negative surface charge on the algae and the algal growth rates which require frequent harvesting compared to terrestrial plants. Algae can be harvested by a number of methods; sedimentation, flocculation, flotation, centrifugation and filtration or a combination of any of these. This paper reviews the various methods of harvesting and dewatering micro-algae for the production of biofuel. There appears to be no one method or combination of harvesting methods suited to all micro-algae and harvesting method will have a considerable influence on the design and operation of both upstream and downstream processes in an overall micro-algal biofuel production process.     

Swimming Using Surface Acoustic Waves

Microactuation of free standing objects in fluids is currently dominated by the rotary propeller, giving rise to a range of potential applications in the military, aeronautic and biomedical fields. Previously, surface acoustic waves (SAWs) have been shown to be of increasing interest in the field of microfluidics, where the refraction of a SAW into a drop of fluid creates a convective flow, a phenomenon generally known as SAW streaming. We now show how SAWs, generated at microelectronic devices, can be used as an efficient method of propulsion actuated by localised fluid streaming. The direction of the force arising from such streaming is optimal when the devices are maintained at the Rayleigh angle. The technique provides propulsion without any moving parts, and, due to the inherent design of the SAW transducer, enables simple control of the direction of travel.     

Acoustical response of hair receptors in insects

Summary A detailed mechanical model is developed to account for the behaviour of hair-like acoustical sensory receptors in insects. For the small hair diameters commonly found, it is concluded that the force acting on the moving hair is caused almost entirely by the viscosity of the air, as analyzed long ago by Stokes. The result of this viscous force is to provide a bending moment about the base of the hair that is proportional to the acoustic particle velocity but that lags behind it by about 135°. In addition the viscous force increases the moment of inertia of the hair by a large and frequency dependent addition, and provides a viscous damping term of sufficient magnitude to reduce the Q value to near unity.The measurements of Tautz (1977) on the thoracic hairs of the caterpillarBarathra brassicae are discussed in detail in terms of the model. Many of these observations are well accounted for, though a few discrepancies remain.This work is part of a programme in biological acoustics supported by the Australian Research Grants Committee.     

An experimental study of the influence of periphytic algae on invertebrate abundance in a Hong Kong stream

1. Small cages (294cm²) containing unglazed clay quarry tiles were used to investigate the influence of periphytic algae on macroinvertebrate abundance in a Hong Kong stream. Algal biomass was manipulated by shading cages with plastic sheets. Individual cages were assigned to one of three treatment groups: unshaded, shaded and deeply shaded. Invertebrate densities and algal biomass within cages were monitored after 23, 37 and 65 days. 2. Multiple-regression analysis revealed that algal biomass, invertebrate morphospecies richness and total abundance declined with greater shading intensity. The responses of individual invertebrate taxa varied: some (especially Trichoptera) were unaffected by shading, whereas grazers (Baetidae, Psephenidae and Elmidae) declined as shading increased. 3. Significant regressions of the densities of individual taxa upon algal and detrital standing stocks in cages had positive slopes, but algal biomass increased during the study while detrital standing stocks declined. Abundance of invertebrates declined or remained rather stable over time. Density increases resulting from a positive association with algae were apparently offset by declines in abundance correlated with reductions in detritus. 4. Declines in algal biomass were associated with greater shading to which animals may respond directly. To uncouple the link between scarcity of algae and reduction of light intensity, the plastic covers on two groups of cages (deeply shaded and unshaded) which had been placed in the stream for 28 days were reversed so that cages which had been shaded became unshaded and vice versa. The cages were recovered on day 33, Only Coleoptera demonstrated a positive association with atgae inside cages; no relationship between population densities and algal biomass or light intensity was apparent for other taxa. However, the design may have been confounded by deposition of sediment in the cages (due to declining stream discharge) which reduced population densities of colonizers. 5. This study documents changes in invertebrate abundance and morphospecies richness in response periphyton and detritus standing stocks within patches. Summation of such responses may account for observed variations in benthic communities among Hong Kong streams which differ in the extent of shading by riparian vegetation.     

On the use of ultrasounds to quantify the longitudinal threshold force to detach osteoblastic cells from a conditioned glass substrate

Cell adhesion on a biomaterial is an important phase of the cell-material interactions and the quality of this phase governs the success of the biomaterial integration. Understanding of the phenomena of cell adhesion and in particular understanding of cell adhesion on biomaterials is of crucial importance for the development of new biomaterials with excellent biocompatibility. One of the physical quantitative indexes to evaluate the quality of cell-material adhesion is its strength. Determining the strength of adhesive bonds requires applying external forces to the cells. Thus, a few methods have been developed to evaluate the strength of cell-material adhesion (micropipette, microplates, microcantilever, ...). These methods apply shear forces on adherent cells. The aim of our work is the development of a new ultrasonic characterization method of cellular adhesion on substrates. With our method, longitudinal acoustic waves are applied on cell culture to impose a longitudinal strain on cells. Only the cells subjected to a sufficient level of strain will be detached from the substrate. The idea is to correlate cell detachment rate to the longitudinal strain threshold supported by cells. From this result, we can deduce the critical force just sufficient to detach the cell. This global method can be adapted for different cell types and for different substrates. This method can provide an evaluation of the effect of functionalization on substrates. The technique is investigated for the 200 kHz ultrasound frequency. An insonificator adapted to the use of cell culture boxes was developed and calibrated. Tests were carried out on a glass substrate with or without biological conditioning. We used the MC3T3-E1 osteoblastic cell line. Our results to date provide the value of the necessary force to detach with reproducibility osteoblastic cells from glass.     

An ultrasonically enhanced inclined settler for microalgae harvesting

Microalgae have vast potential as a sustainable and scalable source of biofuels and bioproducts. However, algae dewatering is a critical challenge that must be addressed. Ultrasonic settling has already been exploited for concentrating various biological cells at relatively small batch volumes and/or low throughput. Typically, these designs are operated in batch or semicontinuous mode, wherein the flow is interrupted and the cells are subsequently harvested. These batch techniques are not well suited for scaleup to the throughput levels required for harvesting microalgae from the large‐scale cultivation operations necessary for a viable algal biofuel industry. This article introduces a novel device for the acoustic harvesting of microalgae. The design is based on the coupling of the acoustophoretic force, acoustic transparent materials, and inclined settling. A filtration efficiency of 70 ± 5% and a concentration factor of 11.6 ± 2.2 were achieved at a flow rate of 25 mL·min^?1 and an energy consumption of 3.6 ± 0.9 kWh·m^?3. The effects of the applied power, flow rate, inlet cell concentration, and inclination were explored. It was found that the filtration efficiency of the device is proportional to the power applied. However, the filtration efficiency experienced a plateau at 100 W L^?1 of power density applied. The filtration efficiency also increased with increasing inlet cell concentration and was inversely proportional to the flow rate. It was also found that the optimum settling angle for maximum concentration factor occurred at an angle of 50 ± 5°. At these optimum conditions, the device had higher filtration efficiency in comparison to other similar devices reported in the previous literature. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:414–423, 2015     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drop-on-demand single cell isolation and total RNA analysis

Technologies that rapidly isolate viable single cells from heterogeneous solutions have significantly contributed to the field of medical genomics. Challenges remain both to enable efficient extraction, isolation and patterning of single cells from heterogeneous solutions as well as to keep them alive during the process due to a limited degree of control over single cell manipulation. Here, we present a microdroplet based method to isolate and pattern single cells from heterogeneous cell suspensions (10% target cell mixture), preserve viability of the extracted cells (97.0±0.8%), and obtain genomic information from isolated cells compared to the non-patterned controls. The cell encapsulation process is both experimentally and theoretically analyzed. Using the isolated cells, we identified 11 stem cell markers among 1000 genes and compare to the controls. This automated platform enabling high-throughput cell manipulation for subsequent genomic analysis employs fewer handling steps compared to existing methods.     

High frequency force generation in outer hair cells from the mammalian ear

Mammalian outer hair cells generate mechanical forces at acoustic frequencies and can thus amplify the sound stimulus within the inner ear. The mechanism of force generation depends upon the plasma membrane potential but not upon either calcium or ATP. Forces are generated in the lateral cortex along the full length of the cell. The cortex includes a two-dimensional cytoskeletal lattice composed of circumferential filaments 6-7 nm thick that are cross-linked by filaments 3-4 nm thick and 40-60 nm long. The two filament types may, respectively, be actin and some form of spectrin. The lattice reinforces the cylindrical shape of the cell and permits limited changes in length. Beneath it lie the lateral cisternae, a regular system of multi-layered membranes. Force-generation may depend upon voltage-dependent shape changes in proteins that lie either in the plasma membrane or in the cytoskeletal lattice.     

The role of stress waves in thoracic visceral injury from blast loading: modification of stress transmission by foams and high-density materials

Materials have been applied to the thoracic wall of anaesthetised experimental animals exposed to blast overpressure to investigate the coupling of direct stress waves into the thorax and the relative contribution of compressive stress waves and gross thoracic compression to lung injury. The ultimate purpose of the work is to develop effective personal protection from the primary effects of blast overpressure--efficient protection can only be achieved if the injury mechanism is identified and characterized. Foam materials acted as acoustic couplers and resulted in a significant augmentation of the visceral injury; decoupling and elimination of injury were achieved by application of a high acoustic impedance layer on top of the foam. In vitro experiments studying stress wave transmission from air through various layers into an anechoic water chamber showed a significant increase in power transmitted by the foams, principally at high frequencies. Material such as copper or resin bonded Kevlar incorporated as a facing upon the foam achieved substantial decoupling at high frequencies--low frequency transmission was largely unaffected. An acoustic transmission model replicated the coupling of the blast waves into the anechoic water chamber. The studies suggest that direct transmission of stress waves plays a dominant role in lung parenchymal injury from blast loading and that gross thoracic compression is not the primary injury mechanism. Acoustic decoupling principles may therefore be employed to reduce the direct stress coupled into the body and thus reduce the severity of lung injury--the most simple decoupler is a high acoustic impedance material as a facing upon a foam, but decoupling layers may be optimized using acoustic transmission models. Conventional impacts producing high body wall velocities will also lead to stress wave generation and transmission--stress wave effects may dominate the visceral response to the impact with direct compression and shear contributing little to the aetiology of the injury.     

Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must be smaller than the incident wavelength of sound and the width of the fluidic channels are typically tens to hundreds of micrometers across, acoustofluidic devices typically use ultrasonic waves generated from a piezoelectric transducer pulsating at high frequencies (in the megahertz range). At characteristic frequencies that depend on the geometry of the device, it is possible to induce the formation of standing waves that can focus particles along desired fluidic streamlines within a bulk flow. Here, we describe a method for the fabrication of acoustophoretic devices from common materials and clean room equipment. We show representative results for the focusing of particles with positive or negative acoustic contrast factors, which move towards the pressure nodes or antinodes of the standing waves, respectively. These devices offer enormous practical utility for precisely positioning large numbers of microscopic entities (e.g., cells) in stationary or flowing fluids for applications ranging from cytometry to assembly.     

Fine structure of the intracochlear potential field. I. The silent current.

Field potentials were recorded along radial tracks in scala tympani and scala vestibuli of the guinea-pig cochlea. A current density analysis revealed standing current density profiles that were qualitatively similar between animals and between the second and third cochlear turns. Radial standing current densities were greatest at or near the spiral ligament. All the scala vestibuli current density profiles were scaled versions of one another while the scala tympani current density profiles showed more variability. Acoustic stimuli modulated the standing current and there was a cochlear microphonic current density peak in scala tympani near the organ of Corti. The results are summarized with a current-density field line model, the key element of which is a constant current pumped into scale media by the stria vascularis. The standing potential gradients drive current from each perilymphatic chamber into the spiral ligament en route to the lateral surface of the stria vascularis. The strial current is divided between the receptor cell pathway and leakage pathways. The standing current through the leakage pathways is indirectly modulated by acoustic stimulation through the modulation of the endocochlear potential. The reciprocal modulation of current between hair cell and leakage pathways suggests that the stria vascularis maintains a constant current during acoustic stimulation. The cochlear standing current is similar to the retinal dark current in its importance for sensory transduction but the fact that the silent current is generated by the stria vascularis and not the receptor cells provides significant benefits for the detection of mechanical stimuli.     

Breakdown of immobilisation/separation and morphology changes of yeast suspended in water-rich ethanol mixtures exposed to ultrasonic plane standing waves

Some physiological/morphological changes have been reported before, when suspended yeasts have been irradiated with well-defined ultrasonic standing, as well as propagating, plane waves around 2.2 MHz, as used in ultrasonic coagulation, e.g., for cell filtering. Thus we used yeast as a biological model to explore the reasons for both those morphology changes and some unusual macroscopic behaviour in the case of water-rich ethanol mixtures when used as carrier liquid. When the cells were suspended in 12% (v/v) ethanol–water mixture separation was greatly reduced; the yeast cells were not retained in the pressure nodal planes of the standing wave, but mixed turbulently through the separation system. How this behaviour alters the efficiency of retention/immobilisation was measured. As the viability of the yeast was decreased as well the morphology of the cells was examined using transmission electron microscopy. Two effects, according to the type of assessment, were evident; a disruption of the cells vacuole and also damage to the cell wall/membrane complex. The extent of the alterations in vacuole structure with sonication time, utilising a fluorescent vacuole membrane dye, was measured. Transient cavitation was not detected and thus could be excluded as being responsible for the observed effects. Other possible reasons for the disruption of the intracellular compartments may be acoustic pressure, displacement or other, secondary effects like (sub) harmonic cavitation. The investigations contribute to a better understanding of the physical conditions experienced when a cell is stressed in a high-frequency ultrasonic wave in the MHz range.