This work was sponsored by the Academic Research Fund (AcRF) Tier 1 (RG171/15), Ministry of Education, Singapore.

1. Preparation of the Microfluidic Channel
<ol>
	<li>Mix poly-dimethylsiloxane (PDMS) with an elastomer base in a ratio of 10:1.</li>
	<li>Degas the mixture in a vacuum oven and pour it on a silicon wafer with a negative tone photoresist pattern on the top.</li>
	<li>Degas the patterned silicon wafer again and heat it at 70 &#176;C for 3 h in an incubator for solidification.</li>
</ol>
2. Fabrication of the Interdigital Transducers
<ol>
	<li>Deposit 20 nm of Cr and 400 nm of Al on a LiNbO3 wafer; pattern 20 strips with a width of 150 &#181;m and an aperture of 2 cm on a plastic mask for photolithography by depositing the positive photoresist on the substrate.</li>
	<li>Remove the Cr-Al layer on the non-exposed area with acetone.</li>
	<li>Treat the surface of them with oxygen plasma (with a nitrogen and oxygen ratio of 2:1) at the power of 30 W for 60 s.</li>
	<li>Align the PDMS microchannel and bond it to the LiNbO3 substrate by pressing it with a thumb for a few seconds.</li>
	<li>Place the integrated device in the heating chamber at 60 &#176;C for 3 h.</li>
</ol>
3. Dual-frequency Excitation
<ol>
	<li>Simultaneously apply two frequency components (f1 and f3, the fundamental frequency and its third harmonic of the fabricated IDT, respectively) with the phase difference &#966; between them to the pair of IDTs, so that the produced SAW may be expressed as follows. 
	<img alt="Equation 1" src="/files/ftp_upload/58085/58085eq1.jpg" /> 
	Here, 
	<img alt="Equation 2" src="/files/ftp_upload/58085/58085eq2.jpg" /> and <img alt="Equation 3" src="/files/ftp_upload/58085/58085eq3.jpg" /> = the acoustic pressures.</li>
	<li>Synthesize the dual-frequency waveform using the Equation Editor of the ArbExpress Application software at the sampling frequency of 100 MS/s and then store it to the function generator as arbitrary input for the SAW excitation in the experiment via a USB cable.</li>
	<li>Vary the power of the fundamental frequency to the total emitted power&#160;<img alt="Equation 4" src="/files/ftp_upload/58085/58085eq4.jpg" /> from 100% (excitation at the purely fundamental frequency) to 0% (excitation at the purely third harmonic); for a good comparison, change but keep the total power the same.</li>
	<li>Vary the phase difference of the dual-frequency excitation from 0&#176; to 360&#176;.</li>
</ol>
4. Numerical Simulation
<ol>
	<li>Describe the motion of the incompressible laminar flow with low Reynolds (i.e., Re = 0.55) and Mach numbers as follows35. 
	<img alt="Equation 5" src="/files/ftp_upload/58085/58085eq5.jpg" /> 
	<img alt="Equation 6" src="/files/ftp_upload/58085/58085eq6.jpg" /> 
	Here, 
	<img alt="Equation 7" src="/files/ftp_upload/58085/58085eq7.jpg" /> = the fluid velocity, 
	<img alt="Equation 8" src="/files/ftp_upload/58085/58085eq8.jpg" /> = the dynamic viscosity, 
	<img alt="Equation 9" src="/files/ftp_upload/58085/58085eq9.jpg" /> = the fluid density, 
	<img alt="Equation 10" src="/files/ftp_upload/58085/58085eq10.jpg" /> = the pressure on the fluid, 
	<img alt="Equation 11" src="/files/ftp_upload/58085/58085eq11.jpg" /> = the identity matrix, and 
	<img alt="Equation 12" src="/files/ftp_upload/58085/58085eq12.jpg" /> = an external force.</li>
	<li>Describe the produced Stoke drag force on the object as follows36. 
	<img alt="Equation 13" src="/files/ftp_upload/58085/58085eq13.jpg" /> 
	Here, 
	<img alt="Equation 14" src="/files/ftp_upload/58085/58085eq14.jpg" />&#160;= the radius of the microparticle, 
	<img alt="Equation 15" src="/files/ftp_upload/58085/58085eq15.jpg" />&#160;= the velocity of the fluid, and 
	<img alt="Equation 16" src="/files/ftp_upload/58085/58085eq16.jpg" />&#160;= the velocity of the microparticle.</li>
	<li>Derive the acoustic radiation force applied to the microparticle in the microchannel along the x-axis (across the microchannel width) at a single frequency as follows16 . 
	<img alt="Equation 17" src="/files/ftp_upload/58085/58085eq17.jpg" /> 
	Here, 
	<img alt="Equation 18" src="/files/ftp_upload/58085/58085eq18.jpg" /> = the volume of the microparticle, 
	<img alt="Equation 19" src="/files/ftp_upload/58085/58085eq19.jpg" /> = the density of the microparticle, 
	<img alt="Equation 20" src="/files/ftp_upload/58085/58085eq20.jpg" /> = the density of the medium, 
	<img alt="Equation 21" src="/files/ftp_upload/58085/58085eq21.jpg" /> = the compressibility of the microparticle, and 
	<img alt="Equation 22" src="/files/ftp_upload/58085/58085eq22.jpg" /> = the compressibility of the medium.</li>
	<li>Derive the resultant acoustic radiation force of the dual-frequency excitation as follows. 
	<img alt="Equation 23" src="/files/ftp_upload/58085/58085eq23.jpg" /></li>
	<li>Express the transverse motion across the channel width (along the y-axis) under both the acoustic radiation force and the Stokes drag force governed by Newton&#39;s second law as follows. 
	<img alt="Equation 24" src="/files/ftp_upload/58085/58085eq24.jpg" /></li>
	<li>Solve the ordinary differential equations (ODEs) above using the fourth order Runge-Kutta method on a personal computer.Set the time step and total duration as 1 &#181;s and 20 s, respectively.</li>
</ol>
5. Experimental Observation
<ol>
	<li>Spin the solution in the concentration of 5.9 x 107 with 4 &#181;m green fluorescent polystyrene beads per 1 mL by vortex for about 2 - 3 min and then immerse it in an ultrasound sonicator for 10 min to disrupt any agglomeration before each testing.</li>
	<li>Fill the mixture into a 3 mL syringe and then drive it with a syringe pump at a flow rate of 3 - 5 &#181;L/min.</li>
	<li>Drive the IDT with the dual-frequency signal from a function generator followed by a power amplifier.</li>
	<li>Observe the stabilized microparticles in the downstream microchannel under a light microscope at a 40X magnification and record the image with a digital camera.</li>
	<li>Measure the location of the accumulated microparticles in the captured digital images using ImageJ and the established scale, and then quantitatively determine the concentration of the accumulated microparticle with the normalized fluorescence brightness at each pressure node.</li>
</ol>

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The authors have nothing to disclose.

The microparticle motion in the microchannel by an SSAW at the dual-frequency excitation was extensively investigated in this study, and an effectively tunable patterning technique by varying the dual-frequency excitation signals was developed and tested. The production of such a waveform is easily realized by most function generators, and the adjusting approach is very convenient. Both the S12- and S11-frequency responses of the fabricated IDTs illustrate several resonant modes34. The measured fundamental frequency of 6.1 MHz and the third harmonic of 17.8 MHz are close to those designed values (6.2 and 18.6 MHz) with similar transmission coefficients, -8.34 dB vs. -9.75 dB, respectively. Thus, a similar acoustic energy output at these two components at the dual-frequency excitation using the single IDT is expected. Such a component combination is not only limited to f1 and f3. Others, such as f1 and f5, and f3 and f5, are also applicable. Although piezoceramics can also generate different harmonics of the bulk acoustic, simultaneous excitation of them is impossible. Switching the acoustic field could enhance the microparticle sorting29 but at the cost of more equipment and a high control complexity.
The number and location of pressure nodes in the microchannel and the corresponding microparticle concentrations could be tuned conveniently and effectively by the dual-frequency excitation without changing other parts. Only one pressure node at P1 &#62; 90% is the same as that produced by the fundamental frequency. However, there are three pressure nodes with varied positions and a microparticle concentration below this threshold. This threshold is found constant for all testing parameters here, such as the driving frequency, the acoustic power, and the diameter of microparticles. The experimental results correlate quite well with the theoretical prediction. By using this proposed strategy, the motion time of microparticles could be reduced up to ~2-fold, which suggests a higher throughput.
Phase modulation with dual frequency provides a flexible control of the pressure nodes location. Shifting other pressure nodes away or adjusting the direction of the acoustic radiation force inward may be a simple way to increase the number of the microparticles on the specific pressure node. At &#966; &#8805; 180&#176;, the pressure node at the bottom of the microchannel will disappear, but the one at the top will show up. At &#966; = 360&#176;, the replacement of pressure nodes occurs. Thus, pressure nodal lines continuously move with the varied phase between two frequency components.
In this study, there are still some limitations. More acoustic attenuation and viscous heating of the wall material may be introduced when a SAW propagates through a thick PDMS microchannel37. Parasitic wave excitation in the wall, such as from a bulk acoustic wave, may also actuate the fluid in the microchannel. Experiments using the biological cells are in a great need for clinical use.
This acoustical LOC is inherently non-invasive, and this new excitation strategy could enhance the tenability and manipulation, which has great potential in many applications. Dual-frequency excitation in biological diagnoses, such as isolating circulating tumor cells (CTCs), may provide information about the occurrence of metastasis and, subsequently, request immediate treatment.

LOC technology integrates one or several functions on a microchip for biology, chemistry, biophysics, and biomedical processes. LOC allows a laboratory set-up on a scale smaller than sub-millimeters, fast reaction rates, a short response time, a high process control, a low volume consumption (less waste, lower reagents cost, and less required sample volume), a high throughput due to parallelization, a low cost in the future mass production and cost-effective disposables, a high safety for chemical, radioactive, or biological studies, and the advantages of a compact and portable device1,2. Precise cell manipulation (i.e., accumulation and separation) is critical in an LOC-based analysis and diagnosis3,4. However, the accuracy and reproducibility of microparticle manipulation have a variety of challenges. Many techniques, such as electro-osmosis5, dielectrophoresis (DEP)6, magnetophoresis7, thermophoresis8,9, an optical approach10, an optoelectronic approach11, a hydrodynamic approach12, and acoustophoresis13,14,15, have been developed. In comparison, acoustic approaches are appropriate for a LOC application because, theoretically, many types of microparticles/cells can be manipulated effectively and noninvasively with a sufficiently high contrast (density and compressibility) compared with the surrounding fluid. Therefore, compared to their counterparts, acoustic approaches are inherently eligible for most microparticles and biological objects, no matter their optical, electrical, and magnetic properties16.
Surface acoustic waves (SAWs) from the IDTs propagate mostly on the surface of a piezoelectric substrate at the thickness of several wavelengths and then leak at the Rayleigh angle into the fluid in the microchannel, according to the Snell&#39;s law17,18,19,20,21,22. They have the technical advantages of a high energy efficiency along the surface due to their localization of the energy, a great design flexibility at high frequency, a good system integration with the microfluidic channel and miniaturization using micro-electronic-mechanical system (MEMS) technology, and a high potential of mass production23. In this protocol, SAWs are generated from a pair of identical IDTs and propagated in the opposite direction to generate a standing wave, or SSAW, in the microchannel, where the suspended microparticles are pushed to pressure nodes, mostly by the applied acoustic radiation force24. The amplitude of such resultant force is determined by the excitation frequency, microparticle size, and its acoustic contrast factor22,25.
Such acoustophoresis has the limitation of predetermined manipulating patterns that are not easily adjustable. The excitation frequency of the IDTs is determined by their periodic distance, so the bandwidth is quite limited. Several strategies have been developed to enhance the tunability and manipulation capability. The first and second modes of acoustic standing waves applied in different parts of the microchannel could separate microparticles more effectively according to different motion speeds toward the nodal lines26. These two modes could also be applied to the whole part of the microchannel and switched alternatively27,28,29. However, for this, a large number of equipment (i.e., three function generators, two impedance matching units, and an electromagnetic relay) is required, with the increased cost and control complexity of the experimental set-up owing to the different electrical impedances at the fundamental frequency and third harmonic of the piezoceramic plate30. Furthermore, slanted-finger interdigital transducers (SFITs) could be applied to adjust the cells and the microparticles patterning by exciting a period of the slanted fingers for a certain resonance20,31. However, then, the bandwidth is inversely proportional to the number of slanted fingers. Multiple pressure nodal lines have a higher separation efficiency and sensitivity in comparison to the single nodal line in the conventional SSAW-based microparticle separator. Alternatively, the location of the pressure nodes could also be changed simply by adjusting the phase difference applied to the two IDTs in the design32,33.
The fundamental frequency and the third harmonic of IDTs have similar frequency responses so that they can be excited simultaneously, which provides more tunability for the microparticles manipulation34. In comparison to the conventional IDT excitation at a single frequency, adjusting the acoustic pressures of the dual-frequency excitation and the phase between them provides technical uniqueness, such as the up to ~2-fold reduced motion time to the pressure nodal line or the center of the microchannel, the varied number and location of the pressure nodal lines, and the microparticle concentrations.

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			<td height="21" style="height:21px;width:257px;">poly-dimethylsiloxane</td>
			<td style="width:147px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">poly-dimethylsiloxane elastomer base</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">silicon wafer</td>
			<td>Bonda Technology</td>
			<td>SI8PSPD</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">negative tone photoresist</td>
			<td>Microchem</td>
			<td>SU-8</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">double-side polished LiNbO3 wafer</td>
			<td>University Wafer</td>
			<td style="width:119px;">Y-128&#176;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">positive photoresist</td>
			<td>Nicolaus-Otto-Stra&#223;e</td>
			<td>AZ 9260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">oxygen plasma</td>
			<td>Harrick Plasma</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic mask</td>
			<td>Infinite Graphics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
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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.

The distributions of the acoustic pressure and the acoustic radiation force of an SSAW at the dual-frequency excitation (6.2 and 18.6 MHz) are shown in Figure 1. Here, the dual-frequency excitation occurs on polystyrene microparticles (4 &#181;m in diameter) in a microchannel with a width of 300 &#181;m at an acoustic power of 146 mW. The resultant acoustic pressure is always in phase when P1 &#62; 90% so that only one pressure node is present at y = 150 &#181;m. In contrast, three pressure nodes are present at y = 75, 150, and 225 &#181;m at P1 = 90%, and at y = 50, 150, and 250 &#181;m at P1 = 0%. The threshold of P1 = 90% is found almost constant throughout all testing conditions, as is a microparticle diameter of 4 - 10 &#181;m, a total acoustic power of 73 - 648 mW, and a driving frequency of 6.2 - 18.6 MHz.
At P1 = 90%, the microparticles in the region of 75 &#181;m &#60; y &#60; 255 &#181;m and 0 &#181;m &#8804; y &#8804; 75 &#181;m move toward the central and the lower pressure node, respectively. In comparison, at P1 = 0%, the regions for the central and the lower pressure nodes are changed to 100 &#181;m &#60; y &#60; 200 &#181;m and 0 &#181;m &#8804; y &#8804; 100 &#181;m, respectively. Subsequently, the microparticle concentrations at the lower node vary from 25% to 33.3%, and at central nodes from 50% to 33.3%, when decreasing P1 from 90% to 0%, respectively (see Figure 2a). The motion time of the microparticle toward the pressure node is reduced from about 1.95 s at P1 = 100% to 0.97 s at P1 = 95% (see Figure 2b). The dependence of the position of the pressure node and the microparticle concentration on P1, measured experimentally, has a good correlation with the numerical prediction (R2 = 0.85 in Figure 2c and R2 = 0.83 in Figure 2d). A large number of power ratios were tested (n &#62; 31), and the variations in the position of accumulated microparticles (6.8 - 10.6%) are much smaller than those in the particle concentration at the pressure nodes (6.7 - 31.4%), which may be due to the occurrence of agglomeration during the microparticle accumulation.
The initial phase of the third harmonic in the dual-frequency excitation affects the synthesized driving waveform, the resultant acoustic radiation force to the microparticle, and the location of the pressure node (see Figure 3). With an increase of &#966; from 0&#176; to 180&#176;, the three pressure nodes (y = 63.5, 150, and 236.5 &#181;m) will gradually shift downward across the microchannel. As P1 was fixed at 85%, the lower pressure node is located at y = 49.5 &#181;m, 33.5 &#181;m, 17 &#181;m, and 0 &#181;m and at &#966; = 45&#176;, 90&#176;, 135&#176;, and 180&#176;, respectively. The acoustic radiation forces from f1 and f3 are out of phase at &#966; = 0&#176;, while in phase at &#966; = 180&#176;. For example, at y = 75 &#181;m and &#966; = 0&#176;, the maximum acoustic radiation forces of f1 and f3 are 37.68 pN and -47.49 pN, respectively. While at &#966; = 180&#176;, the maximum acoustic radiation force from f1 and f3 at the same position are 37.68 pN and 47.49pN, respectively. All pressure nodes shift downward across the microchannel in a linear manner with the increase of &#966;. It is noted that the lower pressure node shifts at a much faster rate than those of the center and the upper pressure nodes (i.e., from 63.5 to 0 &#181;m, from 150 to 110.6 &#181;m, and from 236.5 &#181;m to 190.1 &#181;m with the change of &#966; from 0&#176; to 180&#176;). At &#966; = 180&#176;, there are 4 pressure nodes. After that, the pressure node at the lower boundary (y = 0 &#181;m) disappears, and that at the upper boundary (y = 300 &#181;m) shifts downward at the same rate as the lower pressure node with the change of &#966; from 0&#176; to 180&#176;. At &#966; = 360&#176;, the pressure node replaces an adjacent one (i.e., the upper pressure node at &#966; = 360&#176; has the same location as the central pressure node at &#966; = 0&#176;). The experimental results have a good agreement with the numerical prediction, especially those of the location of the pressure node at different phases.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58085/58085fig1.jpg" /> 
Figure 1. (a) Schematic diagram of experimental setup and (b) photo of IDTs and PDMS microchannel (scale of 300 &#181;m). (c) The pressure waveform and (d) the corresponding acoustic radiation force applied to the 4-&#181;m microspheres in a 300-&#181;m microfluidic channel by the dual-frequency excitation at the varied power ratios of P1 = 100% (purely fundamental frequency), 95%, 91%, 90%, 85%, and 0% (purely third harmonic) at the total acoustic power of 146 mW. Motion of microparticle initially at y = 0 &#181;m (e) with the diameter of 4 &#181;m under the varied power ratios (88-91%) and total acoustic powers (73-648 mW) and (f) with the varied diameters of 4, 6, 8, and 10 &#181;m at the total acoustic power of 73 mW. This figure has been modified from Sriphutkiat, Y., et al.34. <a href="https://www.jove.com/files/ftp_upload/58085/58085fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58085/58085fig2.jpg" /> 
Figure 2. (a) Microparticle position and concentration, (b) motion of microparticles initially at y0 = 0 &#956;m, and the microparticle accumulation time using the dual-frequency excitation at the total acoustic power of 146 mW with varied power ratios. Comparison of the simulation and experimental results (mean standard deviation) of (c) the position of pressure node (R2 = 0.85, n = 37) and (d) the microparticle concentration at each pressure node in the microchannel (R2 = 0.83, n = 31) at the varied power ratios of P1. This figure has been modified from Sriphutkiat, Y., et al.34. <a href="https://www.jove.com/files/ftp_upload/58085/58085fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58085/58085fig3.jpg" /> 
Figure 3. (a) The synthesized waveform at the dual-frequency excitation, (b) the distribution of resultant acoustic radiation force across the 300-&#181;m microchannel at the varied initial phase from 0&#176; to 180&#176; at the power ratio of 85%. The effects of the initial phase at the dual-frequency excitation, &#216;, on the location of pressure node in the (c) simulation and (e) experiment (mean &#177; standard deviation) and the percentage of microparticles accumulated at each pressure node in the (d) simulation and (f) experiment. <a href="https://www.jove.com/files/ftp_upload/58085/58085fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations at JoVE.com

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

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 ...

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6.9K Views.  Nanyang Technological University. A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

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

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 ...

Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments

In materials science and engineering it is often necessary to obtain quantitative measurements of surface topography with micrometer lateral resolution. From the measured surface, 3D topographic maps can be subsequently analyzed using a variety of software packages to extract the information that is needed.
In this article we describe how white light interferometry, and optical profilometry (OP) in general, combined with generic surface analysis software, can be used for materials science and engineering tasks. In this article, a number of applications of white light interferometry for investigation of surface modifications in mass spectrometry, and wear phenomena in tribology and lubrication are demonstrated. We characterize the products of the interaction of semiconductors and metals with energetic ions (sputtering), and laser irradiation (ablation), as well as ex situ measurements of wear of tribological test specimens.
Specifically, we will discuss:
<ol class="lroman">
	<li>Aspects of traditional ion sputtering-based mass spectrometry such as sputtering rates/yields measurements on Si and Cu and subsequent time-to-depth conversion.</li>
	<li>Results of quantitative characterization of the interaction of femtosecond laser irradiation with a semiconductor surface. These results are important for applications such as ablation mass spectrometry, where the quantities of evaporated material can be studied and controlled via pulse duration and energy per pulse. Thus, by determining the crater geometry one can define depth and lateral resolution versus experimental setup conditions.</li>
	<li>Measurements of surface roughness parameters in two dimensions, and quantitative measurements of the surface wear that occur as a result of friction and wear tests.</li>
</ol>
Some inherent drawbacks, possible artifacts, and uncertainty assessments of the white light interferometry approach will be discussed and explained.

White light microscope interferometry is an optical, noncontact and quick method for measuring the topography of surfaces. It is shown how the method can be applied toward mechanical wear analysis, where wear scars on tribological test samples are analyzed; and in materials science to determine ion beam sputtering or laser ablation volumes and depths.

In materials science and engineering it is often necessary to obtain quantitative measurements of surface topography with micrometer lateral resolution. ...

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 ...

Selective Area Modification of Silicon Surface Wettability by Pulsed UV Laser Irradiation in Liquid Environment

The wettability of silicon (Si) is one of the important parameters in the technology of surface functionalization of this material and fabrication of biosensing devices. We report on a protocol of using KrF and ArF lasers irradiating Si (001) samples immersed in a liquid environment with low number of pulses and operating at moderately low pulse fluences to induce Si wettability modification. Wafers immersed for up to 4 hr in a 0.01% H2O2/H2O solution did not show measurable change in their initial contact angle (CA) ~75&#176;. However, the 500-pulse KrF and ArF lasers irradiation of such wafers in a microchamber filled with 0.01% H2O2/H2O solution at 250 and 65 mJ/cm2, respectively, has decreased the CA to near 15&#176;, indicating the formation of a superhydrophilic surface. The formation of OH-terminated Si (001), with no measurable change of the wafer&#8217;s surface morphology, has been confirmed by X-ray photoelectron spectroscopy and atomic force microscopy measurements. The selective area irradiated samples were then immersed in a biotin-conjugated fluorescein-stained nanospheres solution for 2 hr, resulting in a successful immobilization of the nanospheres in the non-irradiated area. This illustrates the potential of&#160;the method for selective area biofunctionalization and fabrication of advanced Si-based biosensing architectures. We also describe a similar protocol of irradiation of wafers immersed in methanol (CH3OH) using ArF laser operating at pulse fluence of 65 mJ/cm2 and in situ formation of a strongly hydrophobic surface of Si (001) with the CA of 103&#176;. The XPS results indicate ArF laser induced formation of Si&#8211;(OCH3)x compounds responsible for the observed hydrophobicity. However, no such compounds were found by XPS on the Si surface irradiated by KrF laser in methanol, demonstrating the inability of the KrF laser to photodissociate methanol and create -OCH3 radicals.

We report on a process of in situ alteration of HF treated Si (001) surface into a hydrophilic or hydrophobic state by irradiating samples in microfluidic chambers filled with&#160;H2O2/H2O solution (0.01%-0.5%) or methanol solutions using pulsed UV laser of a relative low pulse fluence.

The wettability of silicon (Si) is one of the important parameters in the technology of surface functionalization of this material and fabrication of ...

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 ...

Emission Spectroscopic Boundary Layer Investigation during Ablative Material Testing in Plasmatron

Ablative Thermal Protection Systems (TPS) allowed the first humans to safely return to Earth from the moon and are still considered as the only solution for future high-speed reentry missions. But despite the advancements made since Apollo, heat flux prediction remains an imperfect science and engineers resort to safety factors to determine the TPS thickness. This goes at the expense of embarked payload, hampering, for example, sample return missions. 
Ground testing in plasma wind-tunnels is currently the only affordable possibility for both material qualification and validation of material response codes. The subsonic 1.2MW Inductively Coupled Plasmatron facility at the von Karman Institute for Fluid Dynamics is able to reproduce a wide range of reentry environments. This protocol describes a procedure for the study of the gas/surface interaction on ablative materials in high enthalpy flows and presents sample results of a non-pyrolyzing, ablating carbon fiber precursor. With this publication, the authors envisage the definition of a standard procedure, facilitating comparison with other laboratories and contributing to ongoing efforts to improve heat shield reliability and reduce design uncertainties.
The described core techniques are non-intrusive methods to track the material recession with a high-speed camera along with the chemistry in the reactive boundary layer, probed by emission spectroscopy. Although optical emission spectroscopy is limited to line-of-sight measurements and is further constrained to electronically excited atoms and molecules, its simplicity and broad applicability still make it the technique of choice for analysis of the reactive boundary layer. Recession of the ablating sample further requires that the distance of the measurement location with respect to the surface is known at all times during the experiment. Calibration of the optical system of the applied three spectrometers allowed quantitative comparison. At the fiber scale, results from a post-test microscopy analysis are presented.

Development of new ablative materials and their numerical modeling requires extensive experimental investigation. This protocol describes procedures for material response characterization in plasma flows with the core techniques being non-intrusive methods to track the material recession along with the chemistry in the reactive boundary layer by emission spectroscopy.

Ablative Thermal Protection Systems (TPS) allowed the first humans to safely return to Earth from the moon and are still considered as the only solution ...

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 ...

Hand Controlled Manipulation of Single Molecules via a Scanning Probe Microscope with a 3D Virtual Reality Interface

Considering organic molecules as the functional building blocks of future nanoscale technology, the question of how to arrange and assemble such building blocks in a bottom-up approach is still open. The scanning probe microscope (SPM) could be a tool of choice; however, SPM-based manipulation was until recently limited to two dimensions (2D). Binding the SPM tip to a molecule at a well-defined position opens an opportunity of controlled manipulation in 3D space. Unfortunately, 3D manipulation is largely incompatible with the typical 2D-paradigm of viewing and generating SPM data on a computer. For intuitive and efficient manipulation we therefore couple a low-temperature non-contact atomic force/scanning tunneling microscope (LT NC-AFM/STM) to a motion capture system and fully immersive virtual reality goggles. This setup permits &#34;hand controlled manipulation&#34; (HCM), in which the SPM tip is moved according to the motion of the experimenter&#39;s hand, while the tip trajectories as well as the response of the SPM junction are visualized in 3D. HCM paves the way to the development of complex manipulation protocols, potentially leading to a better fundamental understanding of nanoscale interactions acting between molecules on surfaces. Here we describe the setup and the steps needed to achieve successful hand-controlled molecular manipulation within the virtual reality environment.

We demonstrate the precise manipulation of individual organic molecules on a metal surface with the tip of a scanning probe microscope driven in 3D by the experimenter's hand using a motion capture system and fully immersive virtual reality goggles.

Considering organic molecules as the functional building blocks of future nanoscale technology, the question of how to arrange and assemble such building ...

Scanning SQUID Study of Vortex Manipulation by Local Contact

Local, deterministic manipulation of individual vortices in type 2 superconductors is challenging. The ability to control the position of individual vortices is necessary in order to study how vortices interact with each other, with the lattice, and with other magnetic objects. Here, we present a protocol for vortex manipulation in thin superconducting films by local contact, without applying current or magnetic field. Vortices are imaged using a scanning superconducting quantum interference device (SQUID), and vertical stress is applied to the sample by pushing the tip of a silicon chip into the sample, using a piezoelectric element. Vortices are moved by tapping the sample or sweeping it with the silicon tip. Our method allows for effective manipulation of individual vortices, without damaging the film or affecting its topography. We demonstrate how vortices were relocated to distances of up to 0.8 mm. The vortices remained stable at their new location up to five days. With this method, we can control vortices and move them to form complex configurations. This technique for vortex manipulation could also be implemented in applications such as vortex based logic devices.

We present a protocol for manipulation of individual vortices in thin superconducting films, using local mechanical contact. The method does not include applying current, magnetic field or additional fabrication steps.

Local, deterministic manipulation of individual vortices in type 2 superconductors is challenging. The ability to control the position of individual ...

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 ...