Two-photon microscopy of acoustofluidic trapping for highly sensitive cell analysis.

We combine two-photon-excited fluorescence microscopy and acoustofluidic trapping in a spherical microchamber to in vitro study cells and cell clusters three-dimensionally close to in vivo conditions. The two-photon microscopy provides the in-depth 3D analysis of the spherical microchamber dimensions as well as the positions of trapped samples therein with high spatial precision and high temporal resolution enabling even tracking of the fast moving particles. Furthermore, optical sectioning allows to gather information of individual cells in trapped cell clusters inside the chamber. We demonstrate real-time monitoring of osmosis in A549 lung cells and red blood cells as one possible biomedical application. The observed osmosis reduced the cell membrane diameter by approximately 4 µm in the A549 cells and by approximately 2 µm in the red blood cells. Our approach provides an important optical tool for future investigations of cell functions and cell-cell interactions avoiding wall-contact inside an acoustofluidic device.

Rapid measurement of the local pressure amplitude in microchannel acoustophoresis using motile cells.

Acoustic microfluidics (or acoustofluidics) provides a non-contact and label-free means to manipulate and interrogate bioparticles. Owing to their biocompatibility and precision, acoustofluidic approaches have enabled innovations in various areas of biomedical research. Future breakthroughs will rely on the translation of these techniques from academic labs to clinical and industrial settings. Here, accurate characterization and standardization of device performance are crucial. Versatile, rapid, and widely accessible performance quantification is needed. We propose a field quantification method using motile Chlamydomonas reinhardtii algae cells. We previously reported qualitative mapping of acoustic fields using living microswimmers as active probes. In the present study, we extend our approach to achieve the challenging quantitative in situ measurement of the acoustic energy density. C. reinhardtii cells continuously swim in an imposed force field and dynamically redistribute as the field changes. This behavior allows accurate and complete, real-time performance monitoring, which can be easily applied and adopted within the acoustofluidics and broader microfluidics research communities. Additionally, the approach relies only on standard bright-field microscopy to assess the field under numerous conditions within minutes. We benchmark the method against conventional passive-particle tracking, achieving agreement within 1% for field strengths from 0 to 70 J m-3 (from 0 to ∼0.8 MPa).

General defocusing particle tracking.

A General Defocusing Particle Tracking (GDPT) method is proposed for tracking the three-dimensional motion of particles in Lab-on-a-chip systems based on a set of calibration images and the normalized cross-correlation function. In comparison with other single-camera defocusing particle-tracking techniques, GDPT possesses a series of key advantages: it is applicable to particle images of arbitrary shapes, it is intuitive and easy to use, it can be used without advanced knowledge of optics and velocimetry theory, it is robust against outliers and overlapping particle images, and it requires only equipment which is standard in microfluidic laboratories. We demonstrate the method by tracking the three-dimensional motion of 2 µm spherical particles in a microfluidic channel using three different optical arrangements. The position of the particles was measured with an estimated uncertainty of 0.1 µm in the in-plane direction and 2 µm in the depth direction for a measurement volume of 1510 × 1270 × 160 µm(3). A ready-to-use GUI implementation of the method can be acquired on .

Numerical study of acoustophoretic motion of particles in a PDMS microchannel driven by surface acoustic waves.

We present a numerical study of the acoustophoretic motion of particles suspended in a liquid-filled PDMS microchannel on a lithium niobate substrate acoustically driven by surface acoustic waves. We employ a perturbation approach where the flow variables are divided into first- and second-order fields. We use impedance boundary conditions to model the PDMS microchannel walls and we model the acoustic actuation by a displacement function from the literature based on a numerical study of piezoelectric actuation. Consistent with the type of actuation, the obtained first-order field is a horizontal standing wave that travels vertically from the actuated wall towards the upper PDMS wall. This is in contrast to what is observed in bulk acoustic wave devices. The first-order fields drive the acoustic streaming, as well as the time-averaged acoustic radiation force acting on suspended particles. We analyze the motion of suspended particles driven by the acoustic streaming drag and the radiation force. We examine a range of particle diameters to demonstrate the transition from streaming-drag-dominated acoustophoresis to radiation-force-dominated acoustophoresis. Finally, as an application of our numerical model, we demonstrate the capability to tune the position of the vertical pressure node along the channel width by tuning the phase difference between two incoming surface acoustic waves.

Acoustic radiation- and streaming-induced microparticle velocities determined by microparticle image velocimetry in an ultrasound symmetry plane.

We present microparticle image velocimetry measurements of suspended microparticles of diameters from 0.6 to 10 µm undergoing acoustophoresis in an ultrasound symmetry plane in a microchannel. The motion of the smallest particles is dominated by the Stokes drag from the induced acoustic streaming flow, while the motion of the largest particles is dominated by the acoustic radiation force. For all particle sizes we predict theoretically how much of the particle velocity is due to radiation and streaming, respectively. These predictions include corrections for particle-wall interactions and ultrasonic thermoviscous effects and match our measurements within the experimental uncertainty. Finally, we predict theoretically and confirm experimentally that the ratio between the acoustic radiation- and streaming-induced particle velocities is proportional to the actuation frequency, the acoustic contrast factor, and the square of the particle size, while it is inversely proportional to the kinematic viscosity.

A numerical study of microparticle acoustophoresis driven by acoustic radiation forces and streaming-induced drag forces.

We present a numerical study of the transient acoustophoretic motion of microparticles suspended in a liquid-filled microchannel and driven by the acoustic forces arising from an imposed standing ultrasound wave: the acoustic radiation force from the scattering of sound waves on the particles and the Stokes drag force from the induced acoustic streaming flow. These forces are calculated numerically in two steps. First, the thermoacoustic equations are solved to first order in the imposed ultrasound field taking into account the micrometer-thin but crucial thermoviscous boundary layer near the rigid walls. Second, the products of the resulting first-order fields are used as source terms in the time-averaged second-order equations, from which the net acoustic forces acting on the particles are determined. The resulting acoustophoretic particle velocities are quantified for experimentally relevant parameters using a numerical particle-tracking scheme. The model shows the transition in the acoustophoretic particle motion from being dominated by streaming-induced drag to being dominated by radiation forces as a function of particle size, channel geometry, and material properties.

Measuring acoustic energy density in microchannel acoustophoresis using a simple and rapid light-intensity method.

We present a simple and rapid method for measuring the acoustic energy density in microchannel acoustophoresis based on light-intensity measurements of a suspension of particles. The method relies on the assumption that each particle in the suspension undergoes single-particle acoustophoresis. It is validated by the single-particle tracking method, and we show by proper re-scaling that the re-scaled light intensity plotted versus re-scaled time falls on a universal curve. The method allows for analysis of moderate-resolution images in the concentration range encountered in typical experiments, and it is an attractive alternative to particle tracking and particle image velocimetry for quantifying acoustophoretic performance in microchannels.

Automated and temperature-controlled micro-PIV measurements enabling long-term-stable microchannel acoustophoresis characterization.

We present a platform for micro particle image velocimetry (µPIV), capable of carrying out full-channel, temperature-controlled, long-term-stable, and automated µPIV-measurement of microchannel acoustophoresis with uncertainties below 5% and a spatial resolution in the order of 20 µm. A method to determine optimal µPIV-settings for obtaining high-quality results of the spatially inhomogeneous acoustophoretic velocity fields of large dynamical range is presented. In particular we study the dependence of the results on the µPIV interrogation window size and the number of repeated experiments. The µPIV-method was further verified by comparing it with our previously published particle tracking method. Using the µPIV platform we present a series of high-resolution measurements of the acoustophoretic velocity field as a function of the driving frequency, the driving voltage, and the resonator temperature. Finally, we establish a direct and consistent connection between the obtained acoustophoretic velocity fields, and continuous flow mode acoustophoresis, commonly used in applications.

Measuring the local pressure amplitude in microchannel acoustophoresis.

A new method is reported on how to measure the local pressure amplitude and the Q factor of ultrasound resonances in microfluidic chips designed for acoustophoresis of particle suspensions. The method relies on tracking individual polystyrene tracer microbeads in straight water-filled silicon/glass microchannels. The system is actuated by a PZT piezo transducer attached beneath the chip and driven by an applied ac voltage near its eigenfrequency of 2 MHz. For a given frequency a number of particle tracks are recorded by a CCD camera and fitted to a theoretical expression for the acoustophoretic motion of the microbeads. From the curve fits we obtain the acoustic energy density, and hence the pressure amplitude as well as the acoustophoretic force. By plotting the obtained energy densities as a function of applied frequency, we obtain Lorentzian line shapes, from which the resonance frequency and the Q factor for each resonance peak are derived. Typical measurements yield acoustic energy densities of the order of 10 J/m(3), pressure amplitudes of 0.2 MPa, and Q factors around 500. The observed half wavelength of the transverse acoustic pressure wave is equal within 2% to the measured width w = 377 microm of the channel.