This work was supported by the LabEx CeLyA of the University of Lyon (ANR-10-LABX-0060 / ANR-11-IDEX-0007).

1. Design of the acoustic levitation chamber
<ol>
	<li>Design an optically transparent (PMMA-like) cubic tank (8 cm edge and 2.8 mm thickness per face) with the geometry module of a multiphysics simulation software (Table of Materials).</li>
	<li>Insert a cylindrical surface (&#216; = 35 mm) centered at the bottom of the tank, to model the ultrasonic transducer.</li>
	<li>Set the boundary conditions to zero pressure on each wall with a normal displacement of amplitude 1 &#181;m at the transducer surface.</li>
	<li>Using a Frequency domain module, simulate the Frequency Response Function (FRF) of the tank in the frequency range [10 - 40] kHz, at the three arbitrary locations loc1 = (0.01375, 0.01375, 0.04125), loc2 = (0, 0, 0.0088), and loc3 = (0.021725, 0.023375, 0.00935).</li>
	<li>Adapt the tank size such that one of the acoustic modes of the cavity corresponds to the nominal frequency of the transducer (here 31.2 kHz). The FRF therefore contains one resonance peak close to this frequency, as illustrated in Figure 3.</li>
	<li>Plot the pressure field inside the tank, as shown in Figure 4. The chosen resonance mode must contain at least one pressure antinode in the inner of the container, on which the acoustic bubble will be trapped.</li>
	<li>When designing the tank, design a movable top face with a guiding groove on each edge to tightly close the tank faces. Drill a tiny hole at the top face in order to fill the tank with the liquid medium.</li>
	<li>Place the water tank atop a homemade frame that contains the ultrasound transducer (Langevin-type, 31.2 kHz nominal frequency). Use echographic gel to couple the transducer to the tank bottom wall.</li>
	<li>Place the tank and frame system atop a three-directions displacement table with micrometric screws.</li>
	<li>Fill the tank with microfiltered, demineralized and water (not degassed, volume ~500mL, oxygen saturation of approximatively 8 mg&#183;L-1). 
	&#8203;NOTE: Using not degassed water instead of degassed makes it possible to maintaining stable bubbles for the duration of experiments. Using degassed water will accelerate bubble shrinking due to gas diffusion, even if slightly counterbalanced by rectified (ultrasound-mediated) diffusion.</li>
</ol>
2. Bubble generation and acoustic calibration
<ol>
	<li>Prepare the experimental setup used for laser-induced bubble nucleation, acoustic excitation and high-speed recording (Figure 5a,b,c). The experimental setup consists of (A) the acoustic levitation system, (B) the laser power supply and (C) the laser head, (D) one spherical concave lens, (E) one plano-concave lens and one aspherical lens, (F) the high-speed camera, (G) the continuous light-emitting diode. Later, for measurements of liquid flows (Figure 5d) (H) one continuous wave laser source, (I) a cylindrical plano-concave lens followed by a cylindrical plano-concave lens inserted behind the first lens and oriented on the orthogonal axis will be added.</li>
	<li>Plug the ultrasound transducer to a function generator. Set the excitation signal as: sinusoidal waveform, continuous wave, frequency 31.2 kHz. The amplitude is the only variable parameter.</li>
	<li>Place the lens (D) at a distance of approximatively 6 cm in front of the laser head (C).</li>
	<li>Place the lens (E) at a distance of approximatively 12 cm in front of the lens (D).</li>
	<li>Place the water tank (A) such that a focusing point of the laser is located inside the water tank, leading to spark generation for every laser pulse (5 -10 mJ). The laser spark should be located approximately 3 cm below the targeted pressure antinode. 
	NOTE: Without ultrasound (US), laser-nucleated bubble will rise to the top face due to buoyancy.</li>
	<li>Switch on the ultrasound transducer. Increase the applied voltage until the bubble no longer rises vertically but is deviated towards the pressure antinode and, for a sufficient high pressure, trapped.</li>
	<li>Set the backlit illumination (continuous light-emitting diode) and the high-speed camera in order to observe the trapped bubble. 
	NOTE: When nucleating a new bubble with a laser spark, it is easy to capture the trajectory of the bubble approaching its trapping location.</li>
	<li>Move the location of the laser spark within the water tank such that the bubble trajectory remains inside the focal plane of the camera.</li>
	<li>Trap one bubble and capture its radial oscillations with the following parameters: frame size 128 x 128 pixels, acquisition rate 180 kHz. An example of large-amplitude radial oscillations over two acoustic periods is provided in Figure 6. Typical gas bubbles size ranges from 30 to 80 &#181;m.</li>
	<li>Record the bubble radial oscillations during 3 to 30 milliseconds in order to capture hundreds to thousands of bubble oscillations. Repeat this recording for increasing applied voltages of the transducer. Typical applied voltages are in the range of 0 - 8 V. 
	NOTE: When modifying the applied voltage, the equilibrium location of the trapped bubble slightly moves vertically. To follow the oscillations without moving the backlit illumination and camera, place the system (transducer and water tank) onto a three-directions movable table with micrometric accuracy.</li>
	<li>Switch on the ultrasound transducer and capture one image of the background for post-analysis.</li>
	<li>Post-process the video series following this procedure:
	<ol>
		<li>Run the executable file VoltagePressure.exe. The interface shown in Figure 7 should open. 
		NOTE: The script is available as a supplementary document.</li>
		<li>Specify the physical and experimental parameters in the left column (Figure 7A).</li>
		<li>Specify the values of applied voltage for the series of recordings in the bottom-right table (Figure 7B).</li>
		<li>In the Bubble radius analysis panel, click on Load parameters (Figure 7C) and select the folder containing all the files of your video series, and then the background image (mandatory).</li>
		<li>The choice is allowed between analyzing all the videos at once, by clicking on Auto, or one-by-one by clicking on Step by step.</li>
		<li>For each video file, the evolution of the bubble radius is plotted over one acoustic period, and a numerical fit is superimposed. The red curve corresponds to a linearized Rayleigh-Plesset modeling. The equilibrium bubble radius is displayed (Figure 7D).</li>
		<li>According to the numerical fitting, the applied pressure for this voltage is displayed in the Pressure (Voltage) graph panel (Figure 7E). The value of the applied pressure is also displayed in the bottom-right table (Figure 7B). Typical applied pressures corresponding to the 0 - 8 V voltage dynamics are 0 - 25 kPa.</li>
		<li>Once all videos are processed, click on the Linear regression button to perform a linear fit of the pressure/voltage curve. The data (voltage and pressure values) are saved into a .txt file located in the current directory. The slope of the fit is provided.</li>
	</ol>
	</li>
</ol>
3. Coalescence technique
<ol>
	<li>Switch on the ultrasound transducer. Set the applied voltage high enough such that the corresponding acoustic pressure may lead to the triggering of surface instability, according to the numerical pressure/radius diagram of instability zones, as shown in Figure 8.</li>
	<li>Nucleate a bubble, which will then migrate to its trapping location. If the trapped bubble only exhibits spherical oscillations, move on to the next step. If nonspherical oscillations appear:
	<ol>
		<li>Switch off the ultrasound power to let the bubble rise to the top surface.</li>
		<li>Modify the laser energy (by finely tuning over a few mJ) or reduce the transducer voltage.</li>
		<li>Switch on the ultrasound power.</li>
		<li>Nucleate a new bubble.</li>
		<li>Repeat this procedure until the bubble size leads to purely spherical oscillations.</li>
	</ol>
	</li>
	<li>When a trapped bubble exhibits only spherical oscillations, generate a new laser spark. When the new bubble reaches the trapping location, coalescence occurs.</li>
	<li>If the coalesced bubble exhibits only spherical oscillations, generate a new bubble. Multiple coalescences may be necessary to reach the bubble radius at which nonspherical deformations occur. An example of bubble coalescence leading to nonspherical oscillations is shown in Figure 9.</li>
	<li>Once the coalesced bubble exhibits nonspherical oscillations, record the bubble oscillations for a duration of approximatively 3 to 30 milliseconds.</li>
	<li>Identify the mode number of shape oscillations by referring to Figure 10.</li>
</ol>
4. Fluid flow measurements
<ol>
	<li>In the case of cavitation microstreaming measurements, fluorescent tracer particles must be added to the water prior to bubble nucleation. In this study, 0.71 &#181;m particles are used (Table of Materials). They are sufficiently small to be acoustically transparent (not influenced by the acoustic radiation force) and to accurately follow the flow as well as sufficiently large to scatter laser light. Use three drops for the volume of the water tank, corresponding to approximatively 2.104 particles/mm3.</li>
	<li>Prior to taking measurements, set the following parameters for capturing both the (fast timescale) bubble dynamics and (low timescale) fluid flow:
	<ol>
		<li>Create a partitioning of the camera recording disk.</li>
		<li>Alternatively define the recording parameters as:
		<ol>
			<li>Frame rate 180 kHz, frame size 128 x 128 pixels, and exposure time 1 &#181;s for one recording of the dynamics of the bubble interface</li>
			<li>Frame rate 600 Hz, frame size 1024 x 768 pixels, and exposure time 1 ms for one recording of the motion of dye tracers.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Use a continuous laser.</li>
	<li>Create a thin laser sheet by successively letting the laser beam pass through cylindrical plano-concave lens and cylindrical plano-convex lens oriented on an orthogonal axis. A beam width of about 160 &#181;m can be obtained.</li>
	<li>Set up the laser sheet to correspond to the imaging plane:
	<ol>
		<li>Set the laser on a movable device so that the laser sheet can be moved parallel to the imaging plane.</li>
		<li>Adjust the position so that the illuminated particles are visible by the camera.</li>
		<li>Nucleate and trap a bubble.</li>
		<li>Adjust the position of the laser sheet further, so that a shadow becomes visible behind the bubble. The bubble is now inside the laser sheet, as shown in Figure 11.</li>
	</ol>
	</li>
	<li>Induce bubble coalescence until a stably oscillating shape mode is apparent.</li>
	<li>Do several recordings switching back and forth between bubble dynamics and microstreaming. 
	&#8203;NOTE: Switch off the continuous laser when not needed. Heating can create unwanted convective flows. Also, turn off the light-emitting diode when performing the measurements of streaming flow.</li>
</ol>
5. Image processing to visualize the cavitation microstreaming patterns
<ol>
	<li>Install the visualization software ImageJ for image processing and analysis in Java. Also install the plugin CINE File Reader in order to open the high-speed camera files.</li>
	<li>Click on File | Import | CineFile and select the video *.cine containing the capture of the particle motion.</li>
	<li>Select Use virtual stack in the new window, the video is now loaded.</li>
	<li>In order to observe the particle motion without displaying the streaming pattern, click on Image | Adjust | Brightness/Contrast | Auto. The dark background is now replaced by an automatically optimized image.</li>
	<li>In order to display the resulting pattern, click on Image | Stacks | Z Project and choose the Max Intensity option for the image projection. An output image with pixels containing the maximum value over all images in the stack is displayed. Adjust the image contrast as described in step 5.4, if necessary. 
	NOTE: A streaming pattern such as the ones shown in Figure 12b and Figure 12d is obtained.</li>
</ol>

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G., Versluis, M., de Jong, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonspherical+vibrations+of+microbubbles+in+contact+with+a+wall+-+a+pilot+study+at+low+mechanical+index.">Nonspherical vibrations of microbubbles in contact with a wall - a pilot study at low mechanical index.</a> Ultrasound in Medicine and Biology. 34 (4), 685-688 (2008).</li><li>Regnault, G., Mauger, C., Blanc-Benon, P., Inserra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+radiation+force+between+two+closely+spaced+acoustic+bubbles.">Secondary radiation force between two closely spaced acoustic bubbles.</a> Physical Review E. 102, 031101 (2020).</li></ol>

The authors have nothing to disclose.

The presented procedure consists of using bubble coalescence in order to trigger steady-state, symmetry-controlled bubble shape oscillations, allowing the study of the long-term fluid flow induced by these oscillations. The main challenge in the technique is the control of nonspherical oscillations for a bubble being trapped, far from any boundaries.
Most of the existing techniques proposed in the literature focused on substrate-attached bubbles7,<sup class="...

In medicine, an administered drug must penetrate many obstacles in the living system before reaching the desired targets. However, most drugs are rapidly cleaned away from the blood stream. The targeting efficiency is low and they cannot easily cross cell membranes, leading to ineffective drug delivery. Currently, the use of microbubbles in combination with ultrasound has been proposed as an innovative method for noninvasive, precise and targeted delivery of drugs and genes to pathological tissues and cells1. In this approach, microbubbles can play a role as carriers where free drugs are either co-injected with a gas bubble suspension or loaded on its surface. Microbubbles can also act as a local vector for refocusing the ultrasound energy in order to interact with the cells. Basically, under ultrasound exposure, bubbles stably compress and expand, a regime called stable cavitation that generates liquid flows and hence shear stress on nearby objects. Microbubbles may also oscillate non-linearly and expand until collapse, in the regime of inertial cavitation, producing shock waves that propagate radially from the collapse site2. It has been shown that cavitation, either stable or inertial, enhances the permeabilization of cell membranes, and thus enhances the internalization of drugs into the cell3.
In therapeutic applications, understanding the mechanism of the bubble-cell interaction is very important, but there are several barriers, both from scientific and technical sides, that prevent our knowledge from advancing. First, capturing the dynamics of cells in response to bubble-induced mechanical stimuli is very difficult4. At the acoustic timescale, the first-order microbubble oscillations can lead to activation of membrane channels, facilitating molecular passage across biological interfaces. This occurs through the direct oscillation of the cell membrane, also called &#34;cellular massage&#34;5. Channel activation following direct mechanical stress was evidenced using patch-clamp techniques that measured electrophysiological properties of cell membranes during and after ultrasound exposure6. Measuring bubble-induced cell dynamics (meaning the complete field of deformation of the cell membrane) at the acoustic timescale, would also provide insights in the threshold of membrane area expansion &#916;A/A required to induce pores into the cell membrane7. The second barrier is controlling the collapsing bubble regime to avoid microbubble-induced cell lysis. Bubble collapses and induced microjets have been identified as a mechanism through which membrane perforation occurs8,9. Once permeabilized, the cell membrane repairs through calcium self-sealing of the lipid bilayers and fusion of intracellular vesicles9. The occurrence of bubble collapses may also cause lethal damages to the cell and induce unnecessary side effects in the surrounding ones. In sensitive applications such as ultrasound-mediated blood-brain barrier opening, it is generally accepted that inertial bubble collapses should be avoided10.
Therefore, huge efforts are currently devoted to the design of ultrasound emission sequences, coupled with passive cavitation monitoring and control, in order to ensure stable oscillations of microbubbles11. In this stable regime, it has been hypothesized that stably oscillating bubbles play a strong role in the triggering of membrane permeabilization by promoting spatially-targeted shear stress on the cell membrane7. The shear stress results from the liquid flows created in the vicinity of the oscillating bubbles. These liquid flows are called cavitation microstreaming, and, as mentioned above, they are one of the several possible mechanisms which are responsible for enhanced uptake of extracellular molecules. When dealing with suspension of bubbles or cells such as in-vitro biological transfections assays12, permeabilization by microstreaming might be much more efficient than permeabilization by bubble collapse. This can be shown by a simple geometrical consideration. In cell suspensions, sonoporation will be efficient if the majority of the suspended cells is submitted to sufficiently large mechanical effects (leading to membrane permeabilization). It is known that bubble collapses are directed along the direction of isotropic symmetry breaking, such as the bubble-wall axis13 or the bubble-bubble and bubble-cell line joining their center of mass14. The produced microjet is therefore a spatially-localized phenomenon along a finite number of lines joining the cell and bubble centers. Depending on the cell and bubble concentration, as well as the bubble-cell distance, this effect may not be the most efficient to permeabilize the whole number of suspended cells. In contrast, cavitation microstreaming is a phenomenon occurring at a slow timescale, with a large spatial expansion in comparison to the bubble radius. Also, the liquid flow is distributed all around the bubble, and may therefore impacts a larger number of cells, at a very long range. Therefore, understanding the generated cavitation microstreaming around an oscillating bubble is a prerequisite for controlling and quantifying the bubble-induced shear stress that is applied to cells.
To do so, a preliminary step consists in controlling the spherical and nonspherical oscillations of an ultrasound-driven bubble, as the generated liquid flows are induced by the motion of the bubble interface15,16. In particular, shape oscillations of microbubbles have to be triggered and kept stable. Furthermore, the orientation of the bubble shape oscillations has to be controlled to properly analyze the correlation between the bubble interface dynamics and the induced microstreaming pattern. When summarizing the existing literature, it is obvious that detailed experimental results of cavitation-induced microstreaming are only available for bubbles attached to a surface. Wall-attached microbubbles are commonly-used for assessing accurate interface dynamics and cell interactions at the micrometer scale under an ultrafast microscopy system. This configuration is therapeutically relevant when considering vibrating microbubbles located on the cell membrane17,18,19. The study of substrate-attached bubble may however make the analysis of bubble dynamics more complicated, partly due to the complex nature of contact line dynamics20, and the triggering of asymmetric shape modes21. In medical and biological applications, bubbles that are not attached to a wall are commonly found in confined geometries such as small vessels. This impacts significantly bubble dynamics and shape instabilities. Particularly, the presence of a nearby wall shifts the pressure threshold for shape mode triggering to lower pressure values depending on the shape mode number and bubble size22. The wall also affects the bubble-induced microstreaming with possibly higher intensity for the produced flow23.
Amongst all the possible scenario that microbubbles may experience (free or attached, close to a wall, collapsing or stably-oscillating), we propose to investigate the nonspherical dynamics of a single bubble far from any boundary. The experimental setup is based on an acoustic levitation system24 in which a standing ultrasound wave is used to trap the bubble. This scenario is consistent with medical applications in which a collection of suspended bubbles and cells coexist in a sonotransfection chamber, for instance. As far as bubbles and cells are not too close, it is assumed that the presence of a cell does not impact the bubble interface dynamics. When cells follow the loop-like trajectories of the cavitation-induced microstreaming, they are cyclically approaching and repelling from the bubble location and we can assume that the cell presence impacts neither the streaming pattern nor its mean velocity. In addition, nonspherical dynamics and induced microstreaming from single bubbles far from boundary are well known from a theoretical point of view. In order to link the bubble-induced liquid flow to the bubble contour dynamics, it is required to accurately characterize the bubble interface dynamics. To do so, it is preferable to adapt the spatiotemporal scale in experimental studies with respect to those used in therapeutics so that acquisition with common high-speed cameras (below 1 million frame/second) is possible by using large bubbles excited at lower frequencies. When considering uncoated bubbles, the eigenfrequency &#969;n of a given mode n is related to the bubble size as <img alt="Equation 1" src="/files/ftp_upload/62044/62044eq1.jpg" /> 25. This radius-eigenfrequency relationship is slightly modified when considering shelled bubbles26, but the order of magnitude of the eigenfrequency &#969;n remains the same. Thus, investigating bubbles with equilibrium radii ~50&#956;m in a 30 kHz ultrasound field is similar to studying coated bubbles of radii ~3&#956;m in a 1.7 MHz field, as proposed by Dollet et al.27. Similar shape mode numbers and hence microstreaming patterns are therefore expected.
In order to trigger nonspherical oscillations of the bubble interface, it is necessary to exceed a certain pressure threshold that is radius-dependent, as shown in Figure 1. Existing experimental techniques rely on the increase of the acoustic pressure to trigger surface modes (illustrated by path (1) in Figure 1), either by step-by-step pressure increase28 or by modulated-amplitude excitation responsible of periodic onset and extinction of surface modes29. The main drawbacks of these techniques are (i) a random orientation of the symmetry axis of the surface oscillations that cannot be controlled to be in the imaging plane, (ii) a short lifetime of the bubble shape oscillations that makes the analysis of the induced liquid flows difficult at larger timescales, and (iii) the frequent triggering of unstable shape modes. We propose an alternative technique to cross the pressure threshold at a constant acoustic pressure in the radius/pressure map, as illustrated by the path (2) in Figure 1. To do so, it is required to increase the bubble size such that it will be in the instability zone. Such an increase is performed by a bubble coalescence technique. The coalescence of two, initially spherically-oscillating, microbubbles is exploited to create one single deformed bubble. If the acoustic pressure and bubble size of the coalesced bubble are in the instability zone, surface modes are triggered. We also evidenced that the coalescence technique induces stable shape oscillations in a steady-state regime, as well as a controlled symmetry axis defined by the rectilinear motion of the two approaching bubbles. Because a stable shape oscillation is ensured over minutes, the analysis of bubble-induced fluid flow is possible by seeding the liquid medium with fluorescent microparticles, lighted by a thin laser sheet. Recording the motion of the solid microparticles in the vicinity of the bubble interface allows identifying the pattern of the induced fluid flow30. The overall principle of the triggering of bubble shape oscillations, leading to a time-stable fluid flow, is illustrated in Figure 2.
In the following protocol, we outline the steps required to create stable bubble shape oscillations via the coalescence technique and describe the measurements of fluid flow. This includes the design of the acoustic levitation system, the acoustic calibration, bubble nucleation and the coalescence technique, the measurement of bubble interface dynamics and surrounding fluid flow, and the image processing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aspherical lens</td>
			<td>Thorlabs</td>
			<td>AL4050</td>
			<td>Lens of focus 40 mm</td>
		</tr>
		<tr>
			<td>Continuous wave laser source</td>
			<td>CNI</td>
			<td>MLL6FN</td>
			<td>DPSS laser of wavelength 532nm, energy 400 mW</td>
		</tr>
		<tr>
			<td>Cylindrical plano-concave lens</td>
			<td>Thorlabs</td>
			<td>LJ1277L1-A</td>
			<td>lens of focus -25?4mm</td>
		</tr>
		<tr>
			<td>Cylindrical plano-concave lens</td>
			<td>Thorlabs</td>
			<td>LK1900L1</td>
			<td>lens of focus 250 mm</td>
		</tr>
		<tr>
			<td>Fluorescent particles</td>
			<td>Duke Scientific</td>
			<td>R700</td>
			<td>Red polymer fluorescent microspheres</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Agilent</td>
			<td>HP33120</td>
			<td>Generator of function feeding the ultrasound transducer</td>
		</tr>
		<tr>
			<td>High-speed camera</td>
			<td>Vision Research</td>
			<td>Phantom v12.0</td>
			<td>High-speed recording up to 1 Mfps</td>
		</tr>
		<tr>
			<td>Liquid medium</td>
			<td>Carlo Erba</td>
			<td>Water for analysis</td>
			<td>Demineralized, undegassed water</td>
		</tr>
		<tr>
			<td>Multiphysics software</td>
			<td>Comsol</td>
			<td>None</td>
			<td>Softwate for simulating the acoustic field of the levitation chamber</td>
		</tr>
		<tr>
			<td>Nd:Yag pulsed laser</td>
			<td>New Wave Research</td>
			<td>Solo III-15</td>
			<td>5 ns pulse duration, &#955;=532 nm, 3.5 mm beam diameter, up to 50 mJ</td>
		</tr>
		<tr>
			<td>Plano-concave lens</td>
			<td>Thorlabs</td>
			<td>N-BK7</td>
			<td>lens of focus 125 mm</td>
		</tr>
		<tr>
			<td>Spherical concave lens</td>
			<td>Thorlabs</td>
			<td>N-SF11</td>
			<td>Bi-concave lens of focus -25mm</td>
		</tr>
		<tr>
			<td>Ultrasound transducer</td>
			<td>SinapTec</td>
			<td>Custom-made</td>
			<td>Nominal frequency 31kHz, active area 35mm diameter</td>
		</tr>
		<tr>
			<td>Visualization software</td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td>Software for image processing and analysis in Java</td>
		</tr>
		<tr>
			<td>XY Linear stage</td>
			<td>Newport</td>
			<td>M-406</td>
			<td>Displacement stage with micrometric screw</td>
		</tr>
		<tr>
			<td>Z-axis linear stage</td>
			<td>Edmund Optics</td>
			<td>62-299</td>
			<td>Vertical displacement stage with micrometric screw</td>
		</tr>
	</tbody>
</table>

induction of microstreaming by nonspherical bubble oscillations in an acoustic levitation system

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. Experimental observations suggest that the temporary permeabilization of these barriers may be due to shear stress that is exerted on cell tissues by cavitation microstreaming. Cavitation microstreaming is the generation of vortex flows which arise around oscillating ultrasound microbubbles. To produce such liquid flows, bubble oscillations must deviate from purely spherical oscillations and include either a translational instability or shape modes. Experimental studies of bubble-induced flows and shear stress on nearby surfaces are often restricted in their scope due to the difficulty of capturing shape deformations of microbubbles in a stable and controllable manner. We describe the design of an acoustic levitation chamber for the study of symmetry-controlled nonspherical oscillations. Such control is performed by using a coalescence technique between two approaching bubbles in a sufficiently intense ultrasound field. The control of nonspherical oscillations opens the way to a controlled cavitation microstreaming of a free surface-oscillating microbubble. High-frame rate cameras allow investigating quasi-simultaneously the nonspherical bubble dynamics at the acoustic timescale and the liquid flow at a lower timescale. It is shown that a large variety of fluid patterns may be obtained and that they are correlated to the modal content of the bubble interface. We demonstrate that even the high-order shape modes can create large-distance fluid patterns if the interface dynamics contain several modes, highlighting the potential of nonspherical oscillations for targeted and localized drug delivery.

A complete sequence of bubble coalescence leading to time-stable, symmetry-controlled nonspherical oscillations is presented in Figure 9. The approaching phase of two spherically-oscillating bubbles ends when the thin liquid film between the two bubbles is ruptured. It is worth noting that, at the last stage prior to the coalescence, the bubble interfaces deviate from sphericity. Both bubbles elongate on an ellipsoidal shape along the path of the rectilinear ...

Watch this Scientific Journal Video about Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System at JoVE.com

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. ...

induction-microstreaming-nonspherical-bubble-oscillations-an-acoustic

Research

JoVE Journal

Engineering

2.1K Views.  Université Claude Bernard Lyon 1, Centre Léon Bérard, INSERM, UMR 1032, LabTAU, France. A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

Video: Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

High-intensity  focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To  enhance localized heating and improve thermal ablation in ...

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device.

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we ...

Characterization of Full Set Material Constants and Their Temperature Dependence for Piezoelectric Materials Using Resonant Ultrasound Spectroscopy

During the operation of high power electromechanical devices, a temperature rise is unavoidable due to mechanical and electrical losses, causing the degradation of device performance. In order to evaluate such degradations using computer simulations, full matrix material properties at elevated temperatures are needed as inputs. It is extremely difficult to measure such data for ferroelectric materials due to their strong anisotropic nature and property variation among samples of different geometries. Because the degree of depolarization is boundary condition dependent, data obtained by the IEEE (Institute of Electrical and Electronics Engineers) impedance resonance technique, which requires several samples with drastically different geometries, usually lack self-consistency. The resonant ultrasound spectroscopy (RUS) technique allows the full set material constants to be measured using only one sample, which can eliminate errors caused by sample to sample variation. A detailed RUS procedure is demonstrated here using a lead zirconate titanate (PZT-4) piezoceramic sample. In the example, the complete set of material constants was measured from room temperature to 120 &#176;C. Measured free dielectric constants <img alt="Equation 1" src="/files/ftp_upload/53461/image001.jpg" /> and&#160;<img alt="Equation 2" src="/files/ftp_upload/53461/image002.jpg" /> were compared with calculated ones based on the measured full set data, and piezoelectric constants d15 and d33 were also calculated using different formulas. Excellent agreement was found in the entire range of temperatures, which confirmed the self-consistency of the data set obtained by the RUS.

This protocol describes the procedure of measuring the temperature dependence of the full set material constants of piezoelectric materials using resonant ultrasound spectroscopy (RUS).

During the operation of high power electromechanical devices, a temperature rise is unavoidable due to mechanical and electrical losses, causing the ...

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.

Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must ...

Cooling an Optically Trapped Ultracold Fermi Gas by Periodical Driving

We present a cooling method for a cold Fermi gas by parametrically driving atomic motions in a crossed-beam optical dipole trap (ODT). Our method employs the anharmonicity of the ODT, in which the hotter atoms at the edge of the trap feel the anharmonic components of the trapping potential, while the colder atoms in the center of the trap feel the harmonic one. By modulating the trap depth with frequencies that are resonant with the anharmonic components, we selectively excite the hotter atoms out of the trap while keeping the colder atoms in the trap, generating parametric cooling. This experimental protocol starts with a magneto-optical trap (MOT) that is loaded by a Zeeman slower. The precooled atoms in the MOT are then transferred to an ODT, and a bias magnetic field is applied to create an interacting Fermi gas. We then lower the trapping potential to prepare a cold Fermi gas near the degenerate temperature. After that, we sweep the magnetic field to the noninteracting regime of the Fermi gas, in which the parametric cooling can be manifested by modulating the intensity of the optical trapping beams. We find that the parametric cooling effect strongly depends on the modulation frequencies and amplitudes. With the optimized frequency and amplitude, we measure the dependence of the cloud energy on the modulation time. We observe that the cloud energy is changed in an anisotropic way, where the energy of the axial direction is significantly reduced by parametric driving. The cooling effect is limited to the axial direction because the dominant anharmonicity of the crossed-beam ODT is along the axial direction. Finally, we propose to extend this protocol for the trapping potentials of large anharmonicity in all directions, which provides a promising scheme for cooling quantum gases using external driving.

We present a parametric driving method to cool an ultracold Fermi gas in a crossed-beam optical dipole trap. This method selectively removes high-energy atoms from the trap by periodically modulating the trap depth with frequencies that are resonant with the anharmonic components of the trapping potential.

We present a cooling method for a cold Fermi gas by parametrically driving atomic motions in a crossed-beam optical dipole trap (ODT). Our method employs ...

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation displays and solid-state lighting technologies. The brightness and efficiency of blue QDs-based light-emitting diodes (LEDs) remain inferior to their red and green counterparts, due to the inherently unfavorable energy levels of different colors of light. To solve these problems, a device structure should be designed to balance the injection holes and electrons into the emissive QD layer. Herein, through a simple autoxidation strategy, pure blue QD-LEDs which are highly bright and efficient are demonstrated, with a structure of ITO/PEDOT:PSS/Poly-TPD/QDs/Al:Al2O3. The autoxidized Al:Al2O3 cathode can effectively balance the injected charges and enhance radiative recombination without introducing an additional electron transport layer (ETL). As a result, high color-saturated blue QD-LEDs are achieved with a maximum luminance over 13,000 cd m-2, and a maximum current efficiency of 1.15 cd A-1. The easily controlled autoxidation procedure paves the way for achieving high-performance blue QD-LEDs.

A protocol is presented for fabricating high-performance, pure blue ZnCdS/ZnS-based quantum dots light-emitting diodes by employing an autoxidized aluminum cathode.

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation ...

Near-Infrared Temperature Measurement Technique for Water Surrounding an Induction-heated Small Magnetic Sphere

A technique to measure the temperature of water and non-turbid aqueous media surrounding an induction-heated small magnetic sphere is presented. This technique utilizes wavelengths of 1150 and 1412 nm, at which the absorption coefficient of water is dependent on temperature. Water or a non-turbid aqueous gel containing a 2.0-mm- or 0.5-mm-diameter magnetic sphere is irradiated with 1150 nm or 1412 nm incident light, as selected using a narrow bandpass filter; additionally, two-dimensional absorbance images, which are the transverse projections of the absorption coefficient, are acquired via a near-infrared camera. When the three-dimensional distributions of temperature can be assumed to be spherically symmetric, they are estimated by applying inverse Abel transforms to the absorbance profiles. The temperatures were observed to consistently change according to time and the induction heating power.

A technique utilizing wavelengths of 1150 and 1412 nm to measure the temperature of water surrounding an induction-heated small magnetic sphere is presented.

A technique to measure the temperature of water and non-turbid aqueous media surrounding an induction-heated small magnetic sphere is presented. This ...

Quantitative Analysis of Vacuum Induction Melting by Laser-induced Breakdown Spectroscopy

Vacuum induction melting is a popular method for refining high purity metal and alloys. Traditionally, standard process control in metallurgy involves several steps, include drawing samples, cooling, cutting, transport to the laboratory, and analysis. The whole analysis process requires more than 30 minutes, which hinders on-line process control. Laser-induced breakdown spectroscopy is an excellent on-line analysis method that can satisfy the requirements of vacuum induction melting because it is fast and noncontact and does not require sample preparation. The experimental facility uses a lamp-pumped Q-switched laser to ablate melted liquid steel with an output energy of 80 mJ, a frequency of 5 Hz, a FWHM pulse width of 20 ns, and a working wavelength of 1,064 nm. A multi-channel linear charge coupled device (CCD) spectrometer is used to measure the emission spectrum in real time, with a spectral range from 190 to 600 nm and a resolution of 0.06 nm at a wavelength of 200 nm. The protocol includes several steps: standard alloy sample preparation and an ingredient test, smelting of standard samples and determination of the laser breakdown spectrum, and construction of the elements concentration quantitative analysis curve of each element. To realize the concentration analysis of unknown samples, the spectrum of a sample also needs to be measured and disposed with the same process. The composition of all main elements in the melted alloy can be quantitatively analyzed with an internal standard method. The calibration curve shows that the limit of detection of most metal elements ranges from 20-250 ppm. The concentration of elements, such as Ti, Mo, Nb, V, and Cu, can be lower than 100 ppm, and the concentrations of Cr, Al, Co, Fe, Mn, C, and Si range from 100-200 ppm. The R2 of some calibration curves can exceed 0.94.

During vacuum induction melting, laser-induced breakdown spectroscopy is used to perform real-time quantitative analysis of the main-ingredient elements of a molten alloy.

Vacuum induction melting is a popular method for refining high purity metal and alloys. Traditionally, standard process control in metallurgy involves ...

Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip (LOC) system. The simultaneous excitation of the fundamental frequency and its third harmonic, which is termed as dual-frequency excitation, to a pair of interdigital transducers (IDTs) could generate a new type of standing acoustic waves in a microfluidic channel. Varying the power and the phase in the dual-frequency excitation signals results in a reconfigurable field of the acoustic radiation force applied to the microparticles across the microchannel (e.g., the number and location of the pressure nodes and the microparticle concentrations at the corresponding pressure nodes). This article demonstrates that the motion time of the microparticle to only one pressure node can be reduced ~2-fold at the power ratio of the fundamental frequency greater than ~90%. In contrast, there are three pressure nodes in the microchannel if less than this threshold. Furthermore, adjusting the initial phase between the fundamental frequency and the third harmonic results in different motion rates of the three SSAW pressure nodes, as well as in the percentage of microparticles at each pressure node in the microchannel. There is a good agreement between the experimental observation and the numerical predictions. This novel excitation method can easily and non-invasively integrate into the LOC system, with a wide tenability and only a few changes to the experimental set-up.

A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip ...

Safe Experimentation in Optical Levitation of Charged Droplets Using Remote Labs

The work presents an experiment that allows the study of many fundamental physical processes, such as photon pressure, diffraction of light or the motion of charged particles in electrical fields. In this experiment, a focused laser beam pointing upwards levitate liquid droplets. The droplets are levitated by the photon pressure of the focused laser beam which balances the gravitational force. The diffraction pattern created when illuminated with laser light can help measure the size of a trapped droplet. The charge of the trapped droplet can be determined by studying its motion when a vertically directed electrical field is applied. There are several reasons motivating this experiment to be remotely controlled. The investments required for the setup exceeds the amount normally available in undergraduate teaching laboratories. The experiment requires a laser of Class 4, which is harmful to both skin and eyes and the experiment uses voltages that are harmful.

Optical levitation is a method for levitating micrometer-sized dielectric objects using laser light. Utilizing computers and automation systems, an experiment on optical levitation can be controlled remotely. Here, we present a remotely controlled optical levitation system that is used both for educational and research purposes.

The work presents an experiment that allows the study of many fundamental physical processes, such as photon pressure, diffraction of light or the motion ...