Acoustofluidic medium exchange for preparation of electrocompetent bacteria using channel wall trapping.

Comprehensive integration of process steps into a miniaturised version of synthetic biology workflows remains a crucial task in automating the design of biosystems. However, each of these process steps has specific demands with respect to the environmental conditions, including in particular the composition of the surrounding fluid, which makes integration cumbersome. As a case in point, transformation, i.e. reprogramming of bacteria by delivering exogenous genetic material (such as DNA) into the cytoplasm, is a key process in molecular engineering and modern biotechnology in general. Transformation is often performed by electroporation, i.e. creating pores in the membrane using electric shocks in a low conductivity environment. However, cell preparation for electroporation can be cumbersome as it requires the exchange of growth medium (high-conductivity) for low-conductivity medium, typically performed via multiple time-intensive centrifugation steps. To simplify and miniaturise this step, we developed an acoustofluidic device capable of trapping the bacterium Escherichia coli non-invasively for subsequent exchange of medium, which is challenging in acoustofluidic devices due to detrimental acoustic streaming effects. With an improved etching process, we were able to produce a thin wall between two microfluidic channels, which, upon excitation, can generate streaming fields that complement the acoustic radiation force and therefore can be utilised for trapping of bacteria. Our novel design robustly traps Escherichia coli at a flow rate of 10 µL min-1 and has a cell recovery performance of 47 ± 3% after washing the trapped cells. To verify that the performance of the medium exchange device is sufficient, we tested the electrocompetence of the recovered cells in a standard transformation procedure and found a transformation efficiency of 8 × 105 CFU per µg of plasmid DNA. Our device is a low-volume alternative to centrifugation-based methods and opens the door for miniaturisation of a plethora of microbiological and molecular engineering protocols.

Limitation in obtainable surface roughness of hardened cement paste: 'virtual' topographic experiment based on focussed ion beam nanotomography datasets.

Surface roughness affects the results of nanomechanical tests. The surface roughness values to be measured on a surface of a porous material are dependent on the properties of the naturally occurring pore space. In order to assess the surface roughness of hardened cement paste (HCP) without the actual influence of the usual sample preparation for nanomechanical testing (i.e. grinding and polishing), focussed ion beam nanotomography datasets were utilized for reconstruction of 3D (nanoscale resolution) surface profiles of hardened cement pastes. 'Virtual topographic experiments' were performed and root mean square surface roughness was then calculated for a large number of such 3D surface profiles. The resulting root mean square (between 115 and 494 nm) is considerably higher than some roughness values (as low as 10 nm) reported in the literature. We suggest that thus-analysed root mean square values provide an estimate of a 'hard' lower limit that can be achieved by 'artefact-free' sample preparation of realistic samples of hardened cement paste. To the best of our knowledge, this 'hard' lower limit was quantified for a porous material based on hydraulic cement for the first time. We suggest that the values of RMS below such a limit may indicate sample preparation artefacts. Consequently, for reliable nanomechanical testing of disordered porous materials, such as hardened cement paste, the preparation methods may require further improvement.

Oscillating viscometer--evaluation of a new bedside test.

A viscometer for bedside blood measurements was developed, consisting of an oscillating resonator probe mounted directly into a disposable vacutainer tube for blood withdrawal. It was tested in vitro on blood samples with variable hematocrits (20-60%), increasing fibrinogen concentrations (0-20 g/l), increasing concentrations of an admixed radiographic contrast medium and erythrocyte suspensions in dextran 40 and dextran 70. Results were compared with those obtained with a conventional Couette viscometer. Oscillating viscometry yielded generally higher values than Couette viscometry, and had a good sensitivity for changes in hematocrit with a good correlation between the two methods (r=0.96, p<0.0001). Oscillating viscosity depended on the resonator frequency, it was higher at 3900 Hz than at 215 Hz, suggesting a viscoelastic behavior of blood. Erythrocyte aggregation, induced by increasing fibrinogen concentrations or dextran 70, affected oscillating viscometry. At a high frequency, i.e. a smaller penetration depth of the shear wave, oscillating viscosity tended to decrease, which suggests a depletion of the boundary layer from erythrocytes when they aggregate. At low frequency with a deeper shear wave penetration (about 50 microm), erythrocyte aggregation increased oscillating viscosity. Bedside tests in 17 patients with coronary heart disease and 10 controls confirmed the easy practicability of the test and showed lower oscillating viscosity in these patients despite higher fibrinogen concentrations presumably due to increased erythrocyte aggregation. We conclude that oscillating viscometry is an interesting bedside test, which is capable of providing new information on the biorheology of the erythrocyte-poor boundary layer near the vessel wall.

One sensor acoustic emission localization in plates.

Acoustic emissions are elastic waves accompanying damage processes and are therefore used for monitoring the health state of structures. Most of the traditional acoustic emission techniques use a trilateration approach requiring at least three sensors on a 2D domain in order to localize sources of acoustic emission events. In this paper, we present a new approach which requires only a single sensor to identify and localize the source of acoustic emissions in a finite plate. The method proposed makes use of the time reversal principle and the dispersive nature of the flexural wave mode in a suitable frequency band. The signal shape of the transverse velocity response contains information about the propagated paths of the incoming elastic waves. This information is made accessible by a numerical time reversal simulation. The effect of dispersion is reversed and the original shape of the flexural wave is restored at the origin of the acoustic emission. The time reversal process is analyzed first for an infinite Mindlin plate, then by a 3D FEM simulation which in combination results in a novel acoustic emission localization process. The process is experimentally verified for different aluminum plates for artificially generated acoustic emissions (Hsu-Nielsen source). Good and reliable localization was achieved for a homogeneous quadratic aluminum plate with only one measurement.

Enhanced single-cell printing by acoustophoretic cell focusing.

Recent years have witnessed a strong trend towards analysis of single-cells. To access and handle single-cells, many new tools are needed and have partly been developed. Here, we present an improved version of a single-cell printer which is able to deliver individual single cells and beads encapsulated in free-flying picoliter droplets at a single-bead efficiency of 96% and with a throughput of more than 10 beads per minute. By integration of acoustophoretic focusing, the cells could be focused in x and y direction. This way, the cells were lined-up in front of a 40 µm nozzle, where they were analyzed individually by an optical system prior to printing. In agreement with acoustic simulations, the focusing of 10 µm beads and Raji cells has been achieved with an efficiency of 99% (beads) and 86% (Raji cells) to a 40 µm wide center region in the 1 mm wide microfluidic channel. This enabled improved optical analysis and reduced bead losses. The loss of beads that ended up in the waste (because printing them as single beads arrangements could not be ensured) was reduced from 52% ± 6% to 28% ± 1%. The piezoelectric transducer employed for cell focusing could be positioned on an outer part of the device, which proves the acoustophoretic focusing to be versatile and adaptable.

Inverse finite element characterization of soft tissues.

In this work a tissue aspiration method for the in vivo determination of biological soft tissue material parameters is presented. An explicit axisymmetric finite element simulation of the aspiration experiment is used together with a Levenberg-Marquardt algorithm to estimate the material model parameters in an inverse parameter determination process. An optimal fit of the simulated experiment and the real experiment is sought with the parameter estimation algorithm. Soft biological tissue is modelled as a viscoelastic, non-linear, nearly incompressible, isotropic continuum. Viscoelasticity is accounted for by a quasi-linear formulation. The aspiration method is validated experimentally with a synthetic material. In vivo (intra-operatively during surgical interventions) and ex vivo experiments were performed on human uteri.

A newly designed oscillating viscometer for blood viscosity measurements.

A newly designed type of oscillating viscometer is described. The viscometer consists of either a tube or a rod oscillating at a resonance frequency with amplitudes in the micro- and nanometer range. A fluid flowing through the tube or surrounding the rod damps the torsional oscillations. The increase in the damping depends on the viscosity of the fluid and is used to determine viscosity. It was found that viscosity measurements are feasible during blood flow. This new type of viscometer may be useful to the study of biophysical properties of blood at the wall surface during flow and give new insights into blood flow. The device allows direct viscosity measurement on blood directly as it is drawn from the vein through the tube without any anticoagulant.

Rheological properties of blood as assessed with a newly designed oscillating viscometer.

A newly designed type of oscillating viscometer was tested for blood viscosity measurements. The viscometer consists of a probe (either a tube or a rod) oscillating at a resonance frequency with amplitudes in the micro- and nanometer range. The torsional oscillations are dampened by fluids flowing through the tube or surrounding the rod. The degree of damping depends on the viscosity of the fluid, which allows to measure viscosity. Data obtained with these instruments were compared with those obtained with a conventional Couette viscometer. An increase of erythrocyte aggregation by the addition of dextran 70 in vitro led to the expected increase of viscosity in the Couette viscometer; in the oscillating tube viscometer, however, it remained unchanged, which may be explained by a decreased erythrocyte concentration near the tube wall due to increased aggregation and flow of erythrocytes in the tube center. In ex vivo experiments on blood flowing without anticoagulant directly through the tube viscometer an inverse correlation between viscosity and fibrinogen concentration was found. This is in contrast to actual knowledge and may indicate that high fibrinogen levels have a beneficial rheological effect at the tube or vessel wall. Our data suggest that the new oscillating tube viscometer is an interesting tool, which may contribute to a more comprehensive understanding of blood flow.

Positioning of small particles by an ultrasound field excited by surface waves.

A method for the controlled positioning of small particles in one or two dimensions by an ultrasound field excited by a surface wave is presented. Particles of a diameter between 10 and 100 microm placed on a surface can be concentrated at certain locations and moved over the surface. In other approaches it is possible to let the particle levitate freely in the fluid. However for the use of ultrasonic positioning in for example microassembling it is necessary to move particles over a surface as well as to let them levitate over the surface. Physical principle: A two- or three-dimensional ultrasound field is excited in a fluid filled gap between a rigid surface at the bottom and a vibrating surface of a solid at the top. The height of the gap varies between 0.1 and 2 mm. A one-dimensional sinusoidal vibration of the upper surface excites a two-dimensional ultrasound field in the fluid. Particles that are arbitrarily distributed on the lower surface will be concentrated in lines by the ultrasound field. First the calculation of the field of forces on particles in the fluid layer is presented. Then the dispersion relation of a vibrating plate which is in contact with a fluid on one side is derived. The technical setup will be introduced. Finally the experiments are shown and compared to the theoretical results.

Application of neural networks to quantitative nondestructive evaluation.

This paper concentrates on the quantitative analysis of reflection and transmission characteristics of structural waves in notched beams using neural networks. At one end of a rod a piezoelectric transducer excites a flexural guided wave, which propagates through the structure and is reflected from the notch as well as from the ends of the rod. At another point, a laser interferometer measures the resulting displacement versus time, which is recorded and serves as the digital fingerprint of the experiment. Our aim is to obtain quantitative information about the notch characteristics, i.e. its location and depth, on the basis of these digital recordings. This requires an inverse problem to be solved using neural networks. For the training of the network, synthetically produced wave patterns are used. The results of the corresponding wave experiment simulations compare well with experimental data. For fixed location, the calculated wave patterns could be quantitatively analysed to yield the depth of the notch if it is greater than a twentieth of the diameter. Another network could be trained for the localization with an accuracy of 2 cm for 93% of the training patterns. On the basis of these results a strategy is formulated on how neural networks could be trained to evaluate quantitatively both of the notch characteristics.

Simulation of elastic wave propagation in cylindrical structures including excitation by piezoelectric transducers.

The development and optimization of non-destructive testing procedures usually needs experimental data. As experiments are time-consuming and expensive to conduct, we would like to use numerical data instead. This is admissible, if the simulation describes the physical experiments accurately. A three-dimensional displacement-stress finite-difference model is presented for a piezoelectric transducer coupled to an anisotropic tube. The allocation of the displacement and stress components on a staggered grid leads to a stable scheme. A full piezoelectric model of the transducer is used, including transverse isotropy in the elastic, dielectric, and piezoelectric constants. Similar to an experiment, elastic waves are excited in the corresponding simulation by applying a voltage signal to the electrodes of the piezoelectric transducer. Predictions of the simulation model for a piezoelectric ring transducer coupled to a carbon-fibre-reinforced shell are compared to experimental results to test the validity of the numerical data.

Micro-manipulation of small particles by node position control of an ultrasonic standing wave.

For the controlled positioning of small particles with ultrasound a standing wave in a fluid is used. The standing wave is implemented in a resonator, that consists of a fluid filled tube and two piezoelectric transducers on each end. A one-dimensional model of a piezo-device including the fluid-loading on one side and a backside support is introduced. This model allows the calculation of the transmitted wave as a function of the applied electric voltage and the incident wave. In addition, when an electrical impedance is connected to the piezo-device, the reflection coefficient can be varied in amplitude and phase, so that the parameters of the reflected wave can be controlled completely. The resonator itself, consisting of a piezo-device on each end and the fluid between, is included in the model. Several methods to shift the nodes of the standing wave in the resonator are investigated and the ability to position particles is discussed.

Determination of the material properties of microstructures by laser based ultrasound.

In most applications of MEMS the mechanical properties of the used materials are key parameters for the perfect working of the microsystems. Measuring bulk acoustic waves excited in MEMS structures with ultra-short laser pulses is a powerful method for the accurate and non-destructive evaluation as well as for the characterization of material properties. The pump-probe laser-based acoustic method generates bulk acoustic waves in a thermo-elastic way by absorbing the pump laser pulses. The acoustic waves are partly reflected at any discontinuity of the acoustic impedance. At the surface of the specimen the reflected acoustic pulses cause changes of the optical reflection coefficient, which are measured with the probe laser pulses. Thin membranes are part of numerous microelectromechanical systems (MEMS) like sensors, activators and bulk acoustic wave (BAW) filters for example. The described non-destructive and non-contact method is the right approach for testing such thin and brittle structures like membranes. Results of measurements on freestanding aluminium-silicon nitride multi-layer membranes with total thicknesses in the order of several hundred nanometers are presented and compared with thermo-elastic models and with measurements of the supported case. The measured results are used for the determination of the moduli of the membranes.

Ultrasonic wave propagation in focussing tips with arbitrary geometries.

Pulsed laser acoustic experiments have the advantage of very high temporal resolution. However, the lateral resolution amounts to several wavelengths of light. To improve the lateral resolution a focussing tip in which the mechanical waves are focussed is introduced. The combination of high resolution in time and space domain leads to a new potential time resolved scanning probe method. Therefore several axi-symmetric structures are investigated numerically using a finite difference method. The ultrasonic wave propagation in different tips is discussed. By varying the geometry of the tip, the displacement at the sharp end is maximized. The numerically calculated results are verified experimentally on structures having macroscopic dimensions. Scaling effects are considered in order to translate the results into the microscopic scale where arbitrary geometries are much more challenging to implement.

Virtual reality based surgery simulation for endoscopic gynaecology.

Virtual reality (VR) based surgical simulator systems offer very elegant possibilities to both enrich and enhance traditional education in endoscopic surgery. However, while a wide range of VR simulator systems have been proposed and realized in the past few years, most of these systems are far from able to provide a reasonably realistic surgical environment. We explore the basic approaches to the current limits of realism and ultimately seek to extend these based on our description and analysis of the most important components of a VR-based endoscopic simulator. The feasibility of the proposed techniques is demonstrated on a first modular prototype system implementing the basic algorithms for VR-training in gynaecologic laparoscopy.

Acoustic emission localization in beams based on time reversed dispersion.

The common approach for the localization of acoustic emission sources in beams requires at least two measurements at different positions on the structure. The acoustic emission event is then located by evaluating the difference of the arrival times of the elastic waves. Here a new method is introduced, which allows the detection and localization of multiple acoustic emission sources with only a single, one point, unidirectional measurement. The method makes use of the time reversal principle and the dispersive behavior of the flexural wave mode. Whereas time-of-arrival (TOA) methods struggle with the distortion of elastic waves due to phase dispersion, the method presented uses the dispersive behavior of guided waves to locate the origin of the acoustic emission event. Therefore, the localization algorithm depends solely on the measured wave form and not on arrival time estimation. The method combines an acoustic emission experiment with a numerical simulation, in which the measured and time reversed displacement history is set as the boundary condition. In this paper, the method is described in detail and the feasibility is experimentally demonstrated by breaking pencil leads on aluminum beams and pultruded carbon fiber reinforced plastic beams according to ASTM E976 (Hsu-Nielsen source). It will be shown, that acoustic emissions are successfully localized even on anisotropic structures and in the case of geometrical complexities such as notches, which lead to reflections, and cross sectional changes, which affect the wave speed. The overall relative error in localizing the acoustic emission sources was found to be below 5%.

Acoustic field radiated into a transversely isotropic solid from a small aperture spherical surface.

The acoustic field modelling reported in this paper finds application in the design of a scanning probe tip for measuring the near-surface elastic properties of solids and surface structures at high frequencies and with high spatial resolution. The underlying concept is for a longitudinally polarized pulse to be launched from a spherically-shaped portion of the upper surface of the pyramidal or conical shaped tip, and focused towards the narrow lower end. The change in the reflectivity when the narrow end is brought into contact with a solid will provide a measure of the local frequency dependent compliance of that solid. The calculations assume the material from which the tip is fabricated to be transversely isotropic, with symmetry axis coinciding with the axis of the tip. The main issue addressed in this paper is the role of the curvature of the radiating surface and anisotropy of the medium in determining the focal length and focal spread of the radiated field. Two complementary approaches are taken, firstly the discretization of the equations of motion on an irregular mesh of around 3×10(5) triangular elements and solution using the commercial FE package ABAQUS/Explicit, and secondly an analytical approach based on ray tracing and a Green's function method exploiting the angular spectrum method and stationary phase approximation in its evaluation. Consistency is achieved between these approaches regarding the characteristics of the focal region. With the combination of the two approaches it is thus possible to model the wave field from low frequencies, where the FE method is computationally economical and able to handle complex geometries, to high frequencies, where advantage increasingly lies with ray tracing and the Green's function method.

Strategies for single particle manipulation using acoustic and flow fields.

Acoustic radiation forces have often been used for the manipulation of large amounts of micrometer sized suspended particles. The nature of acoustic standing wave fields is such that they are present throughout the whole fluidic volume; this means they are well suited to such operations, with all suspended particles reacting at the same time upon exposure. Here, this simultaneous positioning capability is exploited to pre-align particles along the centerline of channels, so that they can successively be removed by means of an external tool for further analysis. This permits a certain degree of automation in single particle manipulation processes to be achieved as initial identification of particles' location is no longer necessary, rather predetermined. Two research fields in which applications are found have been identified. First, the manipulation of copolymer beads and cells using a microgripper is presented. Then, sample preparation for crystallographic analysis by positioning crystals into a loop using acoustic manipulation and a laminar flow will be presented.

Active fiber composites for the generation of Lamb waves.

Active fiber composites (AFC) are thin and conformable transducer elements with orthotropic material properties, since they are made of one layer of piezoelectric ceramic fibers. They are suitable for applications in structural health monitoring systems (SHM) with acoustic non-destructive testing methods (NDT). In the presented work the transfer behavior of an AFC as an emitter of transient elastic waves in plate-like structures is investigated. The wave field emitted by an AFC surface bonded on an isotropic plate was simulated with the finite-difference method. The model includes the piezoelectric element and the plate and allows the simulation of the elastic wave propagation. For comparison with the model experiments using a laser interferometer for non-contact measurements of particle velocities at different points around the AFC on the surface of the plate were performed. Transfer functions defined as the ratio of the electric voltage excitation signal and the resulting surface velocity at a specific point are separately determined for the two fundamental Lamb wave modes. In order to take the orthotropic behavior of the AFC into account the transfer functions are determined for several points around the AFC. Results show that the AFC is capable to excite the fundamental symmetric and antisymmetric Lamb wave mode. The antisymmetric mode is mainly radiated in the direction of the piezoelectric fibers, while the symmetric mode is spread over a larger angle. The amplitudes of the emitted waves depend on the frequency of the excitation as well as on the geometric dimensions of the transducer.

3D imaging of microstructure of spruce wood.

Synchrotron radiation phase-contrast X-ray tomographic microscopy (srPCXTM) was applied to observation and identification of the features of spruce anatomy at the cellular lengthscale. The pilot experiments presented in the paper clearly revealed the features of the heartwood of Spruce (Picea abies [L.] Karst.), such as lumina and pits connecting the lumina, with a theoretical voxel size of 0.7 x 0.7 x 0.7 microm(3). The experiments were carried out on microspecimens of heartwood, measuring approximately 200 by 200 micrometers in cross-section. The technique for production and preparation of wood microsamples was developed within the framework of this investigation. The total porosity of the samples was derived and the values of the microstructural parameters, such as the diameters of tracheid, cell wall thicknesses and pit diameters were assessed non-invasively. Microstructural features as thin/small as approximately 1.5 microm were revealed and reconstructed in 3D. It is suggested that the position of sub-voxel-sized features (such as position of tori in the bordered pit pairs) can be determined indirectly using watershed segmentation. Moreover, the paper discusses the practical issues connected with a pipelined phase-contrast synchrotron-based microtomography experiment and the possible future potentials of this technique in the domain of wood science.