We thank Drs. Mikhail Shapiro and Nikita Reznik for fruitful discussions. This work was supported by start-up funds from Western University of Health Sciences and NIH grant R21NS101384.

1. Growing Cells on Acoustically-Transparent Polyester Film
<ol>
	<li>Drill a 12 mm hole size at the bottom of a standard 35 mm culture dish using a vertical press-drill. Move the drill slowly and wear eye protection. Remove pieces of plastic attached to the bottom of the dish using a blade to create a smooth surface on the external side (Figure 2).</li>
	<li>Apply a thin layer of marine-grade epoxy or glue at the external bottom surface of the dish.</li>
	<li>Place a film of polyester (2.5 &#181;m thickness) against the external bottom surface of the dish and press firmly to make sure the epoxy/glue spreads evenly between the film and the thick plastic surface. Gently pull the film in a centrifugal manner with fingers to create a flat surface (Figure 2).</li>
	<li>When the epoxy/glue has dried, briefly rinse-dry the polyester-bottom dish with 95% ethanol and sterilize by placing the dish and the inside surface of its lid under a strong 254 nm UV excitation source. Adjust duration and intensity to deliver a UV dose of approximately 330 mJ cm-2 for complete destruction of most types of micro-organisms. This energy approximately corresponds to a duration of 5 min using a 1,000 &#181;W cm-2 UV illumination.</li>
	<li>Aliquot commercially available extracellular matrix protein mixtures (EMPM) in small tubes (50-100 &#181;L) and store them at -20 &#176;C or less in sterile conditions.</li>
	<li>In a sterile environment (e.g., inside a biosafety cabinet), dilute a frozen stock of EMPM with a desired culture medium to 1:100. Work on ice to prevent EMPM polymerization at room temperature. Quickly apply 100 &#181;L of the medium mixture onto the polyester film. Place the lid back on the dish to maintain sterility.</li>
	<li>Incubate EMPM-coated polyester bottom dishes in a cell culture CO2 incubator at 37 &#176;C for 6-12 h.</li>
	<li>After incubation, aspirate the excess medium and directly seed the surface with cells at the desired density. Work under sterile condition to maintain sterility.</li>
</ol>
2. LIPUS Implementation
<ol>
	<li>Place a water tank underneath the objective of an upright microscope with large working volume and without illumination hardware in the transmission path.</li>
	<li>Using commercially-available optomechanical components, place a sample holder below the objective and a transducer holder underneath the sample holder. For subsequent sample search and ultrasound alignment, mount these two holders on translation stages.
	<ol>
		<li>Place the moving parts and actuators of translation stages either outside the tank or above the water line to avoid water damage. Only use non-corrosive materials such as anodized aluminum or stainless steel for immersed optomechanical components.</li>
	</ol>
	</li>
	<li>Fill the tank with deionized and degassed water before utilizing the immersion transducer. The water line should coincide with the horizontal plane of the sample holder (Figure 3). 
	NOTE: Deionized water prevents electrical coupling in presence of high electric fields. Degassing will also prevent scattering and alterations of acoustic waves. Drain water after each experiment using a pump or valve so that the water line falls below the position of the transducer. Also, replace or filter water frequently and clean-up the water tank as needed to avoid growth of microorganisms.</li>
</ol>
3. Oblique Acoustic Excitation
<ol>
	<li>Using commercially available optomechanical components, orient the transducer in an oblique position with respect to the optical path. This will ensure that any reflected waves will be directed away from the sample (Figure 3 and Figure 4).</li>
</ol>
4. Driving the Transducer
NOTE: Ultrasound transducers convert oscillating electrical energy into mechanical expansion/contraction of a piezoelectric material. This conversion produces energy loss in the form of heat energy. Hence, while transducers do possess a peak input voltage limit, they also possess an electrical power limit to avoid thermal damage to the piezoelectric element: 
<img alt="Equation 1" src="/files/ftp_upload/58781/58781eq1.jpg" /> 
with the duty cycle the relative fraction of time of electrical simulation, P the electrical power (in Watts), Vrms the input root-mean-square voltage (in Volts) of the alternative voltage source and Z the electrical impedance (in Ohms). 
<img alt="Equation 2" src="/files/ftp_upload/58781/58781eq2.jpg" /> 
with Vpp the peak-to-peak input voltage applied to the transducer.
<ol>
	<li>Create a sinusoidal wave form containing the desired frequency, number of cycles per pulse, and pulse repetition frequency using a commercial function generator. However, the relatively high Vpp needed to effectively drive standard ultrasound transducers often requires the addition of a power amplifier to amplify the output (i.e., increase the amplitude of Vpp) of the function generator. 
	NOTE: For example, a transducer&#8217;s manufacturer indicates the power limit for a given transducer is 35 W. Will a sinusoidal peak-to-peak input voltage (Vin) of 500 mV at a duty cycle of 50% and amplified through a 50 dB/100 W amplifier be within the power limit of this transducer?
	<ol>
		<li>To answer this question, calculate the voltage after amplification. For a radio-frequency (RF) power amplifier, the amplification factor (dB) is defined by: 
		<img alt="Equation 3" src="/files/ftp_upload/58781/58781eq3.jpg" style="opacity: 0.9;" /> 
		Thus, the amplified voltage has an amplitude output Vpp (Vpp = Vout) of: <img alt="Equation 4" src="/files/ftp_upload/58781/58781eq4.jpg" /> 
		Using Equations 1 and 2, and using 50 &#937; as electrical impedance, the corresponding power generated by this voltage is: 
		<img alt="Equation 5" src="/files/ftp_upload/58781/58781eq5.jpg" /> 
		&#8203;This stimulation is therefore within the power limit of the transducer.</li>
		<li>Using the example above, calculate the waveform parameters (Vpp, frequency, pulse duration and pulse repetition frequency) that correspond to the power and voltage limits provided by the transducer&#8217;s manufacturer. Make sure to respect these limits to avoid damaging the transducer and other connected instruments.</li>
	</ol>
	</li>
	<li>Choose a function generator that operates within a frequency range compatible with the ultrasound transducer. Adjust the frequency of the function generator to the nominal peak frequency of the transducer.</li>
	<li>Create a sinusoidal voltage pulse of the desired duration and repetition frequency using the burst mode of the function generator. Adjust peak-to-peak voltage to a desired value. Make sure that the pulse duration is shorter than the elapsed time between two consecutive pulses.</li>
	<li>Check that the waveform corresponds to the desired signal by connecting the output of the function generator to the input of an oscilloscope.</li>
	<li>Connect the output of the function generator to the input of a power RF amplifier (Figure 4). Make sure that the stimulation parameters are within the limits of the transducer&#8217;s manufacturer.</li>
</ol>
5. Beam Alignment
<ol>
	<li>Choose a hydrophone that operates with a frequency range and acoustic intensity compatible with the frequency and intensity of the ultrasound transducer.</li>
	<li>Carefully bring the tip of a hydrophone probe into focus within the objective field of view at the position corresponding to the position of the sample (Figure 4).</li>
	<li>Make sure that both probe and transducer are immersed in deionized and degassed water. Do not bump the tip of the hydrophone with any physical object other than water as this will alter its coating and affect the measurement.</li>
	<li>Perform a gross pre-alignment of the transducer by visually positioning its acoustic axis toward the hydrophone probe. Makes sure that the distance between the transducer&#8217;s surface and the hydrophone tip correspond approximately to the transducer&#8217;s focal length.</li>
	<li>Connect the hydrophone output to one of the oscilloscope&#8217;s signal input. Connect the synchronization trigger from the function generator to another oscilloscope input. Visualize both signals simultaneously on the oscilloscope.</li>
	<li>Drive the transducer with few ultrasound cycles at a low duty cycle and low amplitude to avoid damaging the probe. Check with the hydrophone&#8217;s manufacturer safe operation conditions to avoid damaging the hydrophone tip.</li>
	<li>Adjust the s/division knob according to the travel time of ultrasound from the transducer&#8217;s surface to the hydrophone. Look for a hydrophone signal on the oscilloscope after the synchronization trigger.</li>
	<li>Slowly actuate the transducer using a motorized or manual XYZ stage. Leave the transducer into the position that correlates with the maximal hydrophone signal (Figure 4). 
	NOTE: If no signal is detected it is possible that the intensity of the acoustic pulses is too low or that the beam is mis-aligned or scattered by an object. Check regularly that the hydrophone and transducer are visually pre-aligned and that no bubbles or physical object are present in the path except the polyester film. If no signal is still detected, increase the input voltage by a small amount to increase the amplitude of hydrophone signal.</li>
</ol>
6. Determination of Ultrasound Pulse Pressure and Intensity
<ol>
	<li>With the beam aligned, measure the peak-to-peak amplitude of the hydrophone output at the oscilloscope for various voltages driving the transducer. Make sure not to exceed the pressure limit recommended by the hydrophone&#8217;s manufacturer.</li>
	<li>Convert these measurements into pressure and/or acoustic intensity values using the calibration method provided by the hydrophone&#8217;s manufacturer. 
	NOTE: The acoustic intensity can be determined from the pressure and vice versa using the formula: 
	<img alt="Equation 6" src="/files/ftp_upload/58781/58781eq6.jpg" /> 
	with I the acoustic pressure (in W m-2), P the acoustic pressure (in Pa), &#961; the density of propagating material (1,000 kg m-3 for water) and c the speed of sound in propagating medium (for water, c = 1,500 m s-1).</li>
	<li>Create calibration curves using these measurements. 
	NOTE: The pressure vs. voltage and intensity vs. voltage curves have a linear and parabolic shape, respectively.</li>
	<li>Determine the pressure and/or intensity value of a desired driving voltage by using the corresponding calibration curve.</li>
</ol>
7. Calcium-Sensitive/LIPUS Live-Cell Fluorescence Imaging
<ol>
	<li>Replace the cell&#8217;s culture medium with a desired imaging buffer containing 5 &#181;M of a cell-permeant calcium-sensitive dye (e.g., Fluo-4 AM). Incubate the culture dish in a CO2 incubator at 37 &#176;C for 1 h.</li>
	<li>Carefully wash cells with the same buffer free of dye.</li>
	<li>Place the dish in the sample holder. Excite the cells using blue light illumination (490 nm) and adjust excitation intensity and camera exposure to avoid excessive bleaching or pixel saturation.</li>
	<li>Perform time-lapse imaging using desired image acquisition settings. Use an immersion objective for better image quality and with long working distance to reduce undesired reflections (see Figure 4).</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanobiology+of+brain+function.">The mechanobiology of brain function.</a> Nature Reviews: Neuroscience. 13 (12), 867-878 (2012).</li><li>Tyler, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+neuromodulation+with+ultrasound?+A+continuum+mechanics+hypothesis.">Noninvasive neuromodulation with ultrasound? A continuum mechanics hypothesis.</a> Neuroscientist. 17 (1), 25-36 (2011).</li><li>Tufail, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+pulsed+ultrasound+stimulates+intact+brain+circuits.">Transcranial pulsed ultrasound stimulates intact brain circuits.</a> Neuron. 66 (5), 681-694 (2010).</li><li>Tyler, W. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+contactless+high+frequency+ultrasound+microbeam+stimulation+for+determination+of+invasion+potential+of+breast+cancer+cells.">Investigating contactless high frequency ultrasound microbeam stimulation for determination of invasion potential of breast cancer cells.</a> Biotechnology and Bioengineering. 110 (10), 2697-2705 (2013).</li><li>Nakano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+ratiometric+fluorescent+thermometer+with+wide+range+and+rapid+response.">Genetically encoded ratiometric fluorescent thermometer with wide range and rapid response.</a> PloS One. 12 (2), e0172344 (2017).</li><li>Donner, J. S., Thompson, S. A., Kreuzer, M. 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The authors have nothing to disclose.

A main advantage of focused ultrasound is its ability to non-invasively deliver mechanical and/or thermal energy to biological samples with high spatio-temporal precision. Other techniques intended to mechanically stimulate cells usually employ invasive physical probes (e.g., cell-poking) or requires the interaction of high energy laser beams with foreign objects (e.g., optical tweezers). Magnetic heating can heat specific spatial locations inside biological samples but requires the presence of foreign magnetic nanoparticles. On the other hand, precise non-invasive heating of small sample (e.g., cell cultures) in aqueous media is possible using infrared or microwave excitation17,18,19.
Since commercial systems able to perform ultrasound excitation in conjunction with fluorescence microscopy are not available, many biophysicists have created customized systems tailored to their specific applications8,12,13,20. The implementation of such systems can, however, be difficult for non-experts. This article described the basic operation to drive a focused ultrasound beam toward the focal plane of an upright epi-fluorescence microscope objective while limiting acoustic reflections and standing waves artefacts that are produced at mismatch acoustic impedance interfaces. These acoustic artifacts are not usually explicitly taken into consideration in the published literature.
If using an acoustic beam perpendicular to the optical path, the beam will ultimately encounter the liquid-solid interface formed by the front lens of an immersed objective or, if an air objective is being used, the water-air interface above the sample. These interfaces will reflect the acoustic waves back to the sample, producing standing waves (see Figure 1). To mitigate or avoid these reflections, it is recommended to position the transducer with an oblique angle with respect to the optical path.
The use of polyester-bottom culture dishes not only minimize acoustic reflections produced by the thick plastic culture dishes bottom, they also enable the experimentalist to stimulate the sample from underneath with an upright microscope. This is a preferable configuration as compared to an inverted microscope to position an immersed transducer with an oblique orientation with respect to the optical path.
This protocol is designed for use with focused ultrasound transducers, but experimentalist may also use non-focused (planar) ultrasound transducers as well. Note that, since LIPUS is intended for precise stimulation of desired areas within tissue, focused ultrasound transducers are generally preferred for both in vitro and in vivo applications. Acoustic beams produced by planar transducers are also broader, making it more difficult to reduce reflections and other mechanical artefacts.
The focal zone of a single element focused ultrasound transducer depends on many parameters such as the element diameter, the acoustic frequency and the sound velocity of the propagating material. For a standard MHz transducer, the focal area is typically confined in a millimetric or sub-millimetric region. A trade-off exists between the size of the focal zone and the ability of the acoustic beam to penetrate inside tissue without too much loss due to attenuation: the higher the frequency, the smaller the focal zone but the weaker the penetration.
To effectively stimulate cells visualized under a microscope, the acoustic focal zone of the transducer must overlap with the optical focal plane of the objective. To this aim, it is necessary to precisely align the acoustic beam using a commercially available hydrophone. There are two main types of hydrophones: hydrophone needles and fiber optic systems. Both types can be used. During alignment, it is important to make sure that the acoustic intensity is within the pressure limits of the hydrophone and that the frequency of the transducer is within the frequency range of the hydrophone&#39;s sensitivity.
Use the calibration provided by the manufacturer (if available) to convert the voltage amplitude output of the hydrophone into actual acoustic pressure (hydrophones are usually calibrated with a number in V Pa-1 or similar units). Hydrophones also usually have a preferential orientation (directionality) with respect to the acoustic beam, hence it is preferred to position the hydrophone in the same direction of the acoustic axis. If not possible, hydrophones may have a chart of attenuation vs. directional angle, allowing directional post-correction after the measurements have been taken.
Beam alignment can be a frustrating task, especially when operating a transducer with a narrow focal zone. The hydrophone signal should appear with a delay with respect to the pulse driving the transducer. This delay corresponds to the time taken by the ultrasound to travel from the transducer&#39;s surface to the hydrophone probe (in water, sound waves travel at a speed of approximately 1,500 m s-1) (see Figure 4). Note that the delay of RF signals traveling through electrical cables is small, only about 3 ns m-1, which, in the present case, can be safely ignored. A way to test if the beam is well aligned is to calculate the distance between transducer and hydrophone using the delay measured at the oscilloscope and the known sound velocity in water. For instance, a delay of approximately 17 &#181;s is expected for a transducer with a focal length of 25 mm.
If the use of commercial hydrophone is not possible (e.g., unusual transducer frequency with not matching hydrophones or limited space for placing the hydrophone), the acoustic beam can be aligned with the pulse-echo method20. This is usually done by first placing a small reflecting object of a size similar or smaller than the beam diameter of the transducer into the focus within the microscope field of view. The transducer is then used both as acoustic emitter (to send ultrasound pulses) and receiver (to detect the echo from the reflecting object). Note that in this configuration, a dedicated amplifier is necessary to amplify the rather small echo signal coming out of the transducer.
For smooth operation of RF instruments, it is important that all the cables and connections to instruments have matching electrical impedance, otherwise undesired electrical reflections will occur and alter the electrical waveform. This is usually the case with most Bayonet Neill-Concelman (BNC) cables and RF equipment having 50 &#937; impedance. Some oscilloscopes, however, have a high input impedance of 1 M&#937; and thus will reflect a RF signal fed through a 50 &#937; BNC cable. In this case, a simple 50 &#937; feed-through terminator should be inserted between the end of the BNC cable and the oscilloscope&#39;s input to properly terminate the signal without loss.
This method is technically limited by the instrumentation used for delivering ultrasound pulses and to perform fluorescence imaging. For example, a standard single-photon fluorescence microscope would only enable imaging of two-dimensional samples. However, it can perform more complex LIPUS imaging of three-dimensional samples like brain slices or small organs using multi-photon excitation.
Another challenge with LIPUS experiments is to distinguish mechanical vs. thermal effects imparted by acoustic beams. A mechanical displacement oblique to the optical path can be detected if it displaces the image in the plane axis (x,y) or defocuses the sample in the z-axis. These displacements depend on the combined optical resolution of the microscope camera and objective. In addition, as these motions would only occur during sonication, one would need to use a frame rate high enough to synchronize the temporal overlap between camera exposure and pulse duration.
To investigate ultrasound-induced thermal effects, the use conventional physical probes to measure temperature is not recommended because of unavoidable vibrations of the probe. However, the technique described here is well suited to measure temperature changes using genetically-encoded thermosensitive fluorescence reporters or thermosensitive dyes21,22. In the future, as more biocompatible fluorescent reporters become available, this technique will enable the study of ultrasound effects on many other biophysical parameters.

Many diseases require some form of invasive medical intervention. These procedures are often expensive, risky, require recovery periods and thus add a burden to health care systems. Non-invasive therapeutic modalities have the potential to provide safer and cheaper alternatives to conventional surgical procedures. However, current non-invasive approaches such as pharmacotherapy or transcranial magnetic stimulation are often limited by trade-offs between tissue penetration, spatiotemporal resolution and unwanted off-target effects. In this context, a focused ultrasound constitutes a promising non-invasive technology with the potential to manipulate biological functions deep inside tissues with high spatiotemporal accuracy and limited off-target effects.
Focused ultrasound stimulation consists of delivering acoustic energy at precise locations deep inside living organisms. Depending on acoustic pulse parameters, this energy can have a variety of medical uses. For instance, the Food and Drug Administration has approved the use of High-intensity Focused Ultrasound (HiFU) for thermal ablation of prostate tumors, tremor-causing brain regions, uterine fibroids and pain-causing nerve endings in bone metastases1. HiFu-mediated microbubble cavitation is also used to transiently open the blood-brain barrier for the targeted delivery of systemically-administered therapeutics2. The spatial-peak pulse-average intensity (Isppa) and spatial-peak temporal-average intensity (Ispta) used for HiFU applications are typically above several kW cm-2 and produce pulse pressure of several tens of MPa. These intensity values are far above the FDA-approved Isppa and Ispta limits for diagnostic ultrasound, 190 W cm-2 and 720 mW cm-2, respectively3. In contrast, recent studies have shown that non-destructive pulsed ultrasound stimulation that are within or near the range of diagnostic ultrasound intensity limits (LIPUS) can be effective to remotely and safely manipulate neural firing4,5,6,7,8, hormonal secretion9,10 and bioengineered cells11. Yet, the cellular and molecular mechanisms by which cells sense and respond to ultrasound remain unclear, precluding clinical translation of LIPUS. Hence, in the past few years, studies of artificial membranes, cultured cells and animals stimulated with ultrasound have gained momentum to reveal biophysical and physiological processes modulated by LIPUS12,13,14,15.
Sound consists of a vibration propagating through a physical medium. An ultrasound is a sound with a frequency above the human audible range (i.e., above 20 kHz). In a laboratory setting, ultrasound waves are generally produced by piezoelectric transducers that contain a material that vibrates in response to an electrical field oscillating in a specific high-frequency bandwidth. Two types of transducers exist: single element transducers and transducer arrays. Single element piezoelectric transducers possess a curved surface which acts as a focusing lens and hence concentrates acoustic energy into a defined region called the focal zone. Single element transducers are much cheaper and easier to operate than transducer arrays. This article will focus on single element transducers.
The size of the focal zone of a focused single element transducer depends on the geometric properties of the acoustic lens and on its acoustic frequency. To achieve a millimeter-size focal zone with a single element transducer, ultrasound frequencies in the MHz range are generally required. Unfortunately, acoustic waves at such frequency are very rapidly attenuated when propagated in a tenuous medium such as air. Thus, MHz ultrasound waves need to be generated and propagated to the sample in a denser material such as water. This constitutes the first challenge in integrating LIPUS modality to a microscope.
A second challenge is to minimize physical interfaces between materials with different acoustic impedances (which is a product of material density and the acoustic velocity) along the acoustic path. These interfaces can reflect, refract, scatter and absorb acoustic waves, making it difficult to quantify the amount of acoustic energy effectively delivered to a sample. They may also create unwanted mechanical artefacts. For instance, reflections produced perpendicular to acoustic mismatch impedance interfaces create backpropagating waves that interfere with forward-propagating ones. Along the interference path, the waves cancel each other at fixed regions of spaces called nodes and sum up at alternating regions called anti-nodes, creating so-called standing waves (Figure 1). It is important for the experimentalist to be able to control or eliminate these experimental interfaces in vitro as they may not exist in vivo.
Fluorescence measurement of optical reporters is a well-known method to interrogate transparent biological samples in real-time and with no physical disturbance. This approach is thus ideal for LIPUS studies as any physical probes present in the sonicated area will introduce mechanical artefacts. This protocol describes the implementation and operation of LIPUS to a commercial epi-fluorescence microscope.

<table>
	<tbody>
		<tr>
			<td>upright microscope with large working volume</td>
			<td>Thorlabs</td>
			<td>CERNA</td>
		</tr>
		<tr>
			<td>upright microscope with large working volume</td>
			<td>Scientifica</td>
			<td>SliceScope</td>
		</tr>
		<tr>
			<td>optomechanical components</td>
			<td>Thorlabs</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>needle hydrophone</td>
			<td>ONDA Corporation</td>
			<td>HNP/C/R/A/T series + AH/G pre-amplifier</td>
		</tr>
		<tr>
			<td>needle hydrophone</td>
			<td>Precision Acoustics</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>fiber optic hydrophone</td>
			<td>ONDA Corporation</td>
			<td>HFO series</td>
		</tr>
		<tr>
			<td>fiber optic hydrophone</td>
			<td>Precision Acoustics</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>oscilloscope</td>
			<td>Keysight Technology</td>
			<td>DSOX2004A (4-channels 70MHz)</td>
		</tr>
		<tr>
			<td>function generator</td>
			<td>Keysight Technology</td>
			<td>33500B (20MHz single-channel)</td>
		</tr>
		<tr>
			<td>RF power amplifier</td>
			<td>Electronic Navigation Industries (ENI)</td>
			<td rowspan="2">325LA, 525LA, 240L, 350L, A075, 2100L, 3100LA</td>
		</tr>
		<tr>
			<td>RF power amplifier</td>
			<td>Electronics &#38; Innovation (E&#38;I)</td>
		</tr>
		<tr>
			<td>immersion ultrasound transducer</td>
			<td>Olympus</td>
			<td>focused immersion transdcuers</td>
		</tr>
		<tr>
			<td>immersion ultrasound transducer</td>
			<td>Benthowave Instrument</td>
			<td>HiFu transducer BII-76 series</td>
		</tr>
		<tr>
			<td>immersion ultrasound transducer</td>
			<td>Precision Acoustics</td>
			<td>Piezo-ceramic or HiFu transducers</td>
		</tr>
		<tr>
			<td>immersion ultrasound transducer</td>
			<td>Ultrasonic-S-lab</td>
			<td>HiFu transducers made to order</td>
		</tr>
		<tr>
			<td>high-density Matrigel</td>
			<td>Corning</td>
			<td>VWR 80094-330</td>
		</tr>
		<tr>
			<td>Mylar film 2.5 microns</td>
			<td>Chemplex</td>
			<td>CAT.NO:107</td>
		</tr>
	</tbody>
</table>

multiplexing focused ultrasound stimulation with fluorescence microscopy

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely manipulate neural firing, hormonal secretion and genetically-reprogrammed cells. However, the translation of this technology for medical applications is currently hampered by a lack of biophysical mechanisms by which targeted tissues sense and respond to LIPUS. A suitable approach to identify these mechanisms would be to use optical biosensors in combination with LIPUS to determine underlying signaling pathways. However, implementing LIPUS to a fluorescence microscope may introduce undesired mechanical artefacts due to the presence of physical interfaces that reflect, absorb and refract acoustic waves. This article presents a step-by-step procedure to incorporate LIPUS to commercially-available upright epi-fluorescence microscopes while minimizing the influence of physical interfaces along the acoustic path. A simple procedure is described to operate a single-element ultrasound transducer and to bring the focal zone of the transducer into the objective focal point. The use of LIPUS is illustrated with an example of LIPUS-induced calcium transients in cultured human glioblastoma cells measured using calcium imaging.

Figure 5 is an example of LIPUS experiment multiplexed with calcium imaging. Glioblastoma cells (A-172) were grown on EMPM coated polyester film in standard culture medium (supplemented with 10% serum and 1% antibiotics) and incubated with the calcium-sensitive fluorescent reporter Fluo-4 AM. Cells were imaged using a 10X immersion lens and illuminated with a white LED light source and fluorescence light was collected using a standard GFP filter set. LIPUS was applied by manually by driving a 4 MHz transducer with a pulse waveform of 158 V peak-to-peak amplitude, 0.1 ms pulse duration and 10 ms pulse repetition frequency (i.e., 1% duty cycle). These parameters correspond to Isppa = 88 W cm-2 (well below the diagnostic limit of 190 W cm-2)and Ispta = 877 mW cm-2 (slightly above the diagnostic limit of 720 mW cm-2), respectively. The results show this stimulation produced robust calcium elevations (Figure 5B, 5C, 5D).
For short pulse durations (i.e., with no significant heat dissipation during the pulse) and assuming the heat capacity of the sample (i.e., cells grown on polyester film and immersed in aqueous solution) is similar to that of water, the change of temperature (&#8710;Tmax) produced during each pulse can be estimated by16
<img alt="Equation 7" src="/files/ftp_upload/58781/58781eq7.jpg" style="opacity: 0.9;" />&#160;<img alt="Equation 8" src="/files/ftp_upload/58781/58781eq8.jpg" style="opacity: 0.9;" />
with a pulse duration of 0.1 ms and pulse intensity of 88 W cm-2
&#8710;Tmax = 0.12 x 0.0001 x 88 &#8776; 1 m&#176;C
with 1% duty cycle, the small amount of heat deposited at the focal zone during each 0.1 ms pulse is likely removed by thermal conduction during the 9.9 ms spanned between two consecutive pulses. Hence, the robust calcium signals seen in Figure 5B, 5C, 5D are most likely induced by non-thermal mechanism(s).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58781/58781fig1.jpg" /> 
Figure 1: Standing wave formation at a reflecting interface. The presence of an interface between materials with different acoustic impedance reflects an incoming pressure wave (blue) with a wavelength &#955;. Since both waves travel in opposite directions, an oscillating phase shift is established. Top: at this time, the phase shift is 180&#176;, producing destructive interference of the standing wave (green wave). Middle: After the waves have moved a distance corresponding to &#955;/4 with respect to the top panel, the phase shift is null and both waves amplify via constructive interferences, producing a standing wave of higher amplitude. Bottom: After the waves have moved an additional distance of &#955;/2 (hence a total of &#955;/4+&#955;/2 = 3/4 &#955; from the top reference), the phase shift becomes null again, producing a standing wave of high amplitude but with inverse polarity. Note that some positions within the path have a constant null pressure (node, black circles) while other positions constantly oscillate between minimum and maximum pressures (antinodes, green circles). <a href="https://www.jove.com/files/ftp_upload/58781/58781fig1large.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/58781/58781fig2.jpg" /> 
Figure 2: Growing cells on acoustically-transparent polyester film. The figure shows the bottom part of a 35 mm dish with a large 12 mm hole in its center. The hole is subsequently covered with a thin polyester film. The film is firmly glued to the external bottom of the dish using marine grade epoxy. <a href="https://www.jove.com/files/ftp_upload/58781/58781fig2large.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/58781/58781fig3.jpg" /> 
Figure 3: Implementation of an ultrasound set-up to an upright fluorescence microscope. A custom-made water tank is placed under an upright fluorescence microscope without transmitted illumination hardware. A motorized sample holder positioned under the objective is attached to optomechanical components fixed to the vibration table outside the tank. The transducer is positioned underneath the sample and attached to a translation stage affixed to optomechanical components inside the tank. <a href="https://www.jove.com/files/ftp_upload/58781/58781fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58781/58781fig4.jpg" /> 
Figure 4: Schematic diagram of the entire set-up. The set-up includes&#160;an epi-fluorescence upright fluorescence microscope to enable fluorescence imaging of cultured cells. The transducer is shown with an oblique orientation with respect to the optical path. This configuration avoids backward reflection of acoustic waves along the acoustic path, thus preventing standing wave formation and/or repetitive stimulation of the sample with multiple ultrasound echoes. A desired waveform is produced by a function generator which can be manually switched on or electronically triggered by a computer interface (Transistor-Transistor-Logic or Universal Serial Bus). The amplitude and delay of the signal measured by the hydrophone needle (red) can be analyzed using an oscilloscope. <a href="https://www.jove.com/files/ftp_upload/58781/58781fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58781/58781fig5.jpg" /> 
Figure 5: Example of LIPUS-induced calcium signals in human glioblastoma cells A-172. (A) Raw fluorescence image of A-172 cells grown on a polyester-bottom 35 mm culture dish and loaded with a cell-permeant version of the calcium-indicator Fluo-4. The red traces represent cell boundaries automatically identified by a computer program and labeled as regions of interest (ROI). (B) Time course of relative fluorescence change (Ft-F0/F0, or &#916;F/F0) for each ROI during a LIPUS experiment. Images were acquired at a speed of 1 frame per second by a standard CCD camera and LIPUS was applied for 10 s between frames 20 and 30. The LIPUS waveform consists of 100 &#181;sec pulses containing 400 cycles at 4 MHz and repeated every 10 ms for 10 s. (C) Plot showing the time course of the mean &#916;F/F0 calculated for all ROIs (error bars not shown). (D) Plot showing the percentile of ROI exhibiting &#916;F/F0 above a user-defined activation threshold. <a href="https://www.jove.com/files/ftp_upload/58781/58781fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy at JoVE.com

Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts.

By focusing low-intensity ultrasound pulses that penetrate soft tissues, LIPUS represents a promising biomedical technology to remotely and safely ...

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8.1K Views.  Western University of Health Sciences. Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a modality for non-invasive mechanical stimulation of endogenous or engineered cells with high spatial and temporal resolution. This article describes how to implement LIPUS to an epi-fluorescence microscope and how to minimize acoustic impedance mismatch along the ultrasound path to prevent unwanted mechanical artefacts. 

Video: Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy

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 ...

Time Multiplexing Super Resolving Technique for Imaging from a Moving Platform

We propose a method for increasing the resolution of an object and overcoming the diffraction limit of an optical system installed on top of a moving imaging system, such as an airborne platform or satellite. The resolution improvement is obtained in a two-step process. First, three low resolution differently defocused images are being captured and the optical phase is retrieved using an improved iterative Gerchberg-Saxton based algorithm. The phase retrieval allows to numerically back propagate the field to the aperture plane. Second, the imaging system is shifted and the first step is repeated. The obtained optical fields at the aperture plane are combined and a synthetically increased lens aperture is generated along the direction of movement, yielding higher imaging resolution. The method resembles a well-known approach from the microwave regime called the Synthetic Aperture Radar (SAR) in which the antenna size is synthetically increased along the platform propagation direction. The proposed method is demonstrated through laboratory experiment.

A method for overcoming the optical diffraction limit is presented. The method includes a two-step process: optical phase retrieval using iterative Gerchberg-Saxton algorithm, and imaging system shifting followed by repetition of the first step. A synthetically increased lens aperture is generated along the direction of movement, yielding higher imaging resolution.

We propose a method for increasing the resolution of an object and overcoming the diffraction limit of an optical system installed on top of a moving ...

Ultrasound Velocity Measurement in a Liquid Metal Electrode

A growing number of electrochemical technologies depend on fluid flow, and often that fluid is opaque. Measuring the flow of an opaque fluid is inherently more difficult than measuring the flow of a transparent fluid, since optical methods are not applicable. Ultrasound can be used to measure the velocity of an opaque fluid, not only at isolated points, but at hundreds or thousands of points arrayed along lines, with good temporal resolution. When applied to a liquid metal electrode, ultrasound velocimetry involves additional challenges: high temperature, chemical activity, and electrical conductivity. Here we describe the experimental apparatus and methods that overcome these challenges and allow the measurement of flow in a liquid metal electrode, as it conducts current, at operating temperature. Temperature is regulated within &#177;2 &#176;C using a Proportional-Integral-Derivative (PID) controller that powers a custom-built furnace. Chemical activity is managed by choosing vessel materials carefully and enclosing the experimental setup in an argon-filled glovebox. Finally, unintended electrical paths are carefully prevented. An automated system logs control settings and experimental measurements, using hardware trigger signals to synchronize devices. This apparatus and these methods can produce measurements that are impossible with other techniques, and allow optimization and control of electrochemical technologies like liquid metal batteries.

Ultrasound velocimetry is used to study mixing by fluid flow in liquid metal electrodes. The focus of this manuscript is to illustrate the methods used for making precise, spatially-resolved ultrasound measurements while limiting oxidation and controlling and monitoring temperature, applied current, and the heater power being supplied.

A growing number of electrochemical technologies depend on fluid flow, and often that fluid is opaque. Measuring the flow of an opaque fluid is inherently ...

Fabrication of Gate-tunable Graphene Devices for Scanning Tunneling Microscopy Studies with Coulomb Impurities

Owing to its relativistic low-energy charge carriers, the interaction between graphene and various impurities leads to a wealth of new physics and degrees of freedom to control electronic devices. In particular, the behavior of graphene&#8217;s charge carriers in response to potentials from charged Coulomb impurities is predicted to differ significantly from that of most materials. Scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) can provide detailed information on both the spatial and energy dependence of graphene&#39;s electronic structure in the presence of a charged impurity.&#160;The design of a hybrid impurity-graphene device, fabricated using controlled deposition of impurities onto a back-gated graphene surface, has enabled several novel methods for controllably tuning graphene&#8217;s electronic properties.1-8 Electrostatic gating enables control of the charge carrier density in graphene and the ability to reversibly tune the charge2 and/or molecular5 states of an impurity. This paper outlines the process of fabricating a gate-tunable graphene device decorated with individual Coulomb impurities for combined STM/STS studies.2-5 These studies provide valuable insights into the underlying physics, as well as signposts for designing hybrid graphene devices.

This paper details the fabrication process of a gate-tunable graphene device, decorated with Coulomb impurities for scanning tunneling microscopy studies. Mapping the spatially dependent electronic structure of graphene in the presence of charged impurities unveils the unique behavior of its relativistic charge carriers in response to a local Coulomb potential.

Owing to its relativistic low-energy charge carriers, the interaction between graphene and various impurities leads to a wealth of new physics and degrees ...

In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography (CT) and Light Microscopy (LM) Correlated with Scanning Electron Microscopy (SEM)

In failure analysis, device characterization and reverse engineering of light emitting diodes (LEDs), and similar electronic components of micro-characterization, plays an important role. Commonly, different techniques like X-ray computed tomography (CT), light microscopy (LM) and scanning electron microscopy (SEM) are used separately. Similarly, the results have to be treated for each technique independently. Here a comprehensive study is shown which demonstrates the potentials leveraged by linking CT, LM and SEM. In depth characterization is performed on a white emitting LED, which can be operated throughout all characterization steps. Major advantages are: planned preparation of defined cross sections, correlation of optical properties to structural and compositional information, as well as reliable identification of different functional regions. This results from the breadth of information available from identical regions of interest (ROIs): polarization contrast, bright and dark-field LM images, as well as optical images of the LED cross section in operation. This is supplemented by SEM imaging techniques and micro-analysis using energy dispersive X-ray spectroscopy.

In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography CT and Light Microscopy LM Correlated with Scanning Electron Microscopy SEM

A workflow for comprehensive micro-characterization of active optical devices is outlined. It contains structural as well as functional investigations by means of CT, LM and SEM. The method is demonstrated for a white LED which can be still be operated during characterization.

In failure analysis, device characterization and reverse engineering of light emitting diodes (LEDs), and similar electronic components of ...

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution optimization.

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown ...

Focused Ion Beam Fabrication of LiPON-based Solid-state Lithium-ion Nanobatteries for In Situ Testing

Solid-state electrolytes are a promising replacement for current organic liquid electrolytes, enabling higher energy densities and improved safety of lithium-ion (Li-ion) batteries. However, a number of setbacks prevent their integration into commercial devices. The main limiting factor is due to nanoscale phenomena occurring at the electrode/electrolyte interfaces, ultimately leading to degradation of battery operation. These key problems are highly challenging to observe and characterize as these batteries contain multiple buried interfaces. One approach for direct observation of interfacial phenomena in thin film batteries is through the fabrication of electrochemically active nanobatteries by a focused ion beam (FIB). As such, a reliable technique to fabricate nanobatteries was developed and demonstrated in recent work. Herein, a detailed protocol with a step-by-step process is presented to enable the reproduction of this nanobattery fabrication process. In particular, this technique was applied to a thin film battery consisting of LiCoO2/LiPON/a-Si, and has further been previously demonstrated by in situ cycling within a transmission electron microscope.

Focused Ion Beam Fabrication of LiPON-based Solid-state Lithium-ion Nanobatteries for In Situ Testing

A protocol for the fabrication of electrochemically active LiPON-based solid-state lithium-ion nanobatteries using a focused ion beam is presented.

Solid-state electrolytes are a promising replacement for current organic liquid electrolytes, enabling higher energy densities and improved safety of ...

Three-Dimensional Ultrasonic Needle Tip Tracking with a Fiber-Optic Ultrasound Receiver

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. When visualization is lost, the medical device can cause trauma to critical tissue structures. Here, a method to track the needle tip during ultrasound image-guided procedures is presented. This method involves the use of a fiber-optic ultrasound receiver that is affixed within the cannula of a medical needle to communicate ultrasonically with the external ultrasound probe. This custom probe comprises a central transducer element array and side element arrays. In addition to conventional two-dimensional (2D) B-mode ultrasound imaging provided by the central array, three-dimensional (3D) needle tip tracking is provided by the side arrays. For B-mode ultrasound imaging, a standard transmit-receive sequence with electronic beamforming is performed. For ultrasonic tracking, Golay-coded ultrasound transmissions from the 4 side arrays are received by the hydrophone sensor, and subsequently the received signals are decoded to identify the needle tip&#39;s spatial location with respect to the ultrasound imaging probe. As a preliminary validation of this method, insertions of the needle/hydrophone pair were performed in clinically realistic contexts. This novel ultrasound imaging/tracking method is compatible with current clinical workflow, and it provides reliable device tracking during in-plane and out-of-plane needle insertions.

Accurate and efficient visualization of invasive medical devices is extremely important in many ultrasound-guided minimally invasive procedures. Here, a method for localizing the spatial position of a needle tip relative to the ultrasound imaging probe is presented.

Ultrasound is frequently used for guiding minimally invasive procedures, but visualizing medical devices is often challenging with this imaging modality. ...

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 ...

Controllable Nucleation of Cavitation from Plasmonic Gold Nanoparticles for Enhancing High Intensity Focused Ultrasound Applications

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) for the controllable nucleation of cavitation in tissue-mimicking gel phantoms. This in vitro protocol was developed to demonstrate the feasibility of this approach, for both enhancement of imaging and therapeutic applications for cancer. The same apparatus can be used for both imaging and therapeutic applications by varying the exposure duration of the HIFU system. For short duration exposures (10 &#181;s), broadband acoustic emissions were generated through the controlled nucleation of inertial cavitation around the gold nanoparticles. These emissions provide direct localization of nanoparticles. For future applications, these particles may be functionalized with molecular-targeting antibodies (e.g. anti-HER2 for breast cancer) and can provide precise localization of cancerous regions, complementing routine diagnostic ultrasound imaging. For continuous wave (CW) exposures, the cavitation activity was used to increase the localized heating from the HIFU exposures resulting in larger thermal damage in the gel phantoms. The acoustic emissions generated from inertial cavitation activity during these CW exposures was monitored using a passive cavitation detection (PCD) system to provide feedback of cavitation activity. Increased localized heating was only achieved through the unique combination of nanoparticles, laser light and HIFU. Further validation of this technique in pre-clinical models of cancer is necessary.

This protocol demonstrates the controllable nucleation of cavitation in gel phantoms, through simultaneous exposure to both near-infrared pulsed laser light and high intensity focused ultrasound (HIFU). The cavitation activity can then be used for enhancing imaging and/or therapeutic uses of HIFU.

In this study, plasmonic gold nanoparticles were simultaneously exposed to pulsed near-infrared laser light and high intensity focused ultrasound (HIFU) ...