The authors thank the Engineering and Physical Sciences Research Council for supporting this work through grant EP/L024012/1. VB is also supported by the Engineering and Physical Sciences Research Council (EPSRC) and Medical Research Council (MRC) (grant EP/L016052/1). VB and AV thank the Clarendon Foundation for Post Graduate Scholarships. AV also thanks Exeter College for a Santander scholarship. The authors are indebted to James Fisk and David Salisbury for their invaluable assistance in the manufacturing of the apparatus. They also gratefully acknowledge the contributions of Drs. Dario Carugo and Joshua Owen in the development of earlier prototype SATs.

The authors have nothing to disclose.

The critical steps for any acoustic measurement were encapsulated by Apfel in 198176 as &#8220;know thy liquid, know thy sound field, know when something happens.&#8221; In the context of this protocol, these encompass the transducer calibration and alignment and the water preparation and bubble handling steps. First, it is essential that the hydrophone used to calibrate the driving transducer and/or the PCD is itself accurately calibrated through regular external servicing or in-house comparison to a reference standard. Similarly, the response of both the driving transducer and PCD need to be regularly characterized to check for any change in output and/or loss of sensitivity. If the driving conditions and receive sensitivity of the system are unknown, then it will be impossible to infer any meaningful relationship between exposure conditions, bioeffects and acoustic emissions. Directly related to this, the alignment of the transducers to each other and the sample chamber needs to be carefully checked to ensure that the exposure conditions within the chamber are as expected and the sampling volume for the PCD corresponds to the region of interest. As indicated, the temperature and gas content of the suspending medium can affect the final results significantly and consistency is extremely important in this respect77,78. Similarly, the preparation, characterization, and handling of the cavitation agent suspension require very close attention to ensure that the expected size distribution and concentration of particles is present within the sample. For example, if the concentration of bubbles is too high, there will be effective shielding of the sample volume from the incident US field. MB agents are particularly susceptible to destruction and coalescence and further guidance on their handling may be found in Mulvana et. al. (2012)79.
A very common problem with detection of cavitation signals is achieving an adequate SNR. This is due partly to the nature of the signal itself, as described, but may also be due to sources of electrical noise within the experimental set up. Checking the connections between system components, in particular those involving co-axial cables, may help to eliminate some of these. Replacing or repairing co-axial cables may be necessary. Identifying and removing or deactivating other equipment in the laboratory such as pumps that may cause electrical noise can also help. Poor electrical impedance matching between system components can be a further cause of poor signal to noise ratio and also potentially of damage to equipment and should be carefully checked. The triggering settings on the signal generator and oscilloscope should similarly be checked to confirm that they are configured appropriately for the experiment and have not reverted to the manufacturer default settings. If there is significant destruction of bubbles during handling, in case of the SAT2, it may be helpful to attach a second syringe to the outlet port and use this to gently extract fluid from the chamber, thereby drawing in the suspension. This can also help in eliminating macrobubbles or enabling flow during US exposure, if desired.
It is not possible to completely eliminate acoustic reflections within the sample chamber and hence the incident field will not be completely uniform over the whole&#160;sample volume. As mentioned in steps 1.3.2 and 1.3.3, the transmissibility of acoustic windows will be frequency dependent and thus the desired bandwidth for acoustic emission measurements should be carefully considered. In particular, there may be significant multiple reflections of higher frequency components. This is another reason why calibration of the field within the fully assembled system is so important for minimizing the uncertainty in incident pressure. Appropriate gating of the recorded signals should also be considered to minimize the effects of multiple reflections. The use of commercial devices for convenience and the need for acoustic transparency means that some optical transparency must be sacrificed. This may impact the quality of subsequent imaging, e.g., to assess cell viability or drug uptake. Some of the membranes used in commercial devices are also porous and, thus, imperfect isolation occurs between the sample chamber and the surrounding water bath. As above, the corresponding risk of contamination can be mitigated by using a smaller sub-chamber, the contents of which can be regularly replaced. The cell culture devices indicated in the Table of Materials are suitable primarily for cell monolayers that may not be representative of tissues in terms of all US/cavitation-mediated bioeffects. The proximity of the cells to a solid surface will also affect MB dynamics in a way that may not be reflective of conditions in vivo, e.g., promoting microstreaming and microjetting as described in the introduction. These limitations can be addressed, however, through a simple substitution of alternative tissue models.
The aim in proposing the SATs is to provide a means of improving the reproducibility of acoustic exposure conditions and acoustic emissions between studies of US-mediated bioeffects, thus hopefully facilitating better understanding of the underlying mechanisms and the development of treatment monitoring techniques to improve safety and efficacy. The systems are designed to be compatible with commercially available cell culture devices, enabling a wide range of biological assays to be performed according to the application of interest and enabling the performance of high throughput experiments, removing the need for time consuming alignment procedures between runs. By standardizing protocols for the characterization of exposure conditions and the capture of acoustic emissions, the system dependent variability can hopefully be reduced. The range of parameters that should be explored for a particular experiment will depend upon the application (desired bio-effect, cell type, depth of target tissue if in vivo etc.) and the nature of any cavitation agent being used. Given the large number of variables (US frequency, pressure amplitude, pulse length, pulse repetition frequency etc.) fully exploring the whole parameter space is unlikely to be practicable. An advantage of the proposed protocol is that it enables some bounds on this parameter space to be quickly established. For example, it enables determination of the minimum pressure at which a cavitation signal is generated, the maximum pressure or pulse length that can be used before cell detachment/death occurs, and the pressure at which fractional harmonics or broadband noise are produced. It is recommended that such a set of scoping measurements be carried out as a first step in any study.
As presented, the SATs are designed for real-time monitoring of acoustic emissions, with biological assays being performed outside of the experiment. It would be relatively simple, however, to modify the SAT to enable direct optical observation of the sample chamber via a microscope objective. This could in turn be coupled to a fluorescence and/or high-speed microscopy system to enable observation of drug uptake and bubble dynamics, for example. The PCD output as currently presented in terms of voltage indicates: i) the types of cavitation behavior and their relative proportions; ii) how long these cavitation behaviors persist; iii) whether the observed time-cumulative exposure characteristics are correlated to a particular bioeffect; and iv) whether the relative levels and time-dependent behaviors are consistent with previous experiments in the exposure system. Whilst the receive sensitivity of the PCD can be quantified, in order to reliably characterize the acoustic emissions in terms of absolute energy, additional spatial information is required. This could be achieved by replacing the PCD with an array probe to implement passive acoustic mapping (PAM)80. This would, however increase the complexity of signal processing and the computational time and power required.
Other instrumentation for measurement of membrane electrical resistance or application of physical targeting methods, for example magnetic fields, could also be incorporated. It would also be possible to use three-dimensional tissue structures such as tumor spheroids, organoids, or even ex vivo tissue samples on acoustically &#8220;soft&#8221; gel substrates in place of the cell monolayers to study US and cavitation-mediated effects in more realistic tissue environments.

Ultrasound (US) has been used widely as a diagnostic imaging technique because of its safe and non-invasive nature, its ease of implementation at a patient&#8217;s bedside, and its cost effectiveness1. Next to its diagnostic and monitoring capabilities, US has considerable potential for therapeutic applications. Early work explored its use in thrombolysis, DNA transfection, and drug delivery2,3,4 and therapeutic US now represents a very active area of research, with applications including tumor treatment5,6,7, immunotherapy8,9, blood-brain barrier (BBB) disruption10,11,12, thrombolysis13,14,15, and bacterial infection treatment16,17. A key phenomenon underpinning these applications is cavitation: the nucleation, growth, and oscillation of gaseous cavities due to changes in fluid pressure18,19.
There is a range of mechanisms by which cavitation produces biological effects. For example, the highly nonlinear nature of bubble oscillations under the influence of an applied US field can generate microstreaming in the surrounding liquid that can both enhance drug convection20 and exert shear stresses on the tissue in the vicinity of the bubbles. This is particularly prevalent when bubbles are in the vicinity of a boundary, causing bubbles to oscillate non-spherically, and may potentially promote drug uptake through shear-induced permeabilization21,22,23,24. At higher pressures, larger amplitude oscillations and rapid bubble collapse are observed, imparting direct mechanical stress25 and frequently generating shock waves, and consequent large pressure gradients&#160;that can disrupt and permeabilize tissues26,27. The collapse of bubbles near a surface can also result in the formation of high-velocity liquid microjets28,29,30. These microjets can penetrate tissue, potentially creating pores or inducing secondary stress waves31,32. The permeabilization of biological membranes at both the tissue and cellular levels is variously referred to as sonophoresis, used primarily in the context of US-induced enhancement in skin permeability33,34, and sonoporation, used mainly to describe the reversible permeabilization of the cellular membrane due to formation of membrane pores35,36.
Viscous absorption in the liquid immediately surrounding the oscillating bubble can produce a substantial heating effect37. Moreover, the highly non-linear oscillations produce acoustic radiation at frequencies higher than the driving US&#160;field. This leads to increased absorption in the surrounding tissue and further heating38. Bubble collapse may also be accompanied by chemical effects due to the transient high temperatures and pressures in the bubble core, such as generation of highly reactive species and electromagnetic radiation, known as sonoluminescence32. These effects have been investigated to assess potential damage and/or activation of relevant cellular pathways for delivery39 and exploited in local activation of light-sensitive drugs in an approach known as sonodynamic therapy40,41,42,43.
Many US-mediated bioeffects may be initiated solely through control of US field parameters (pressure amplitude, frequency, pulse length and repetition frequency, and duration of exposure), but reliably generating&#160;cavitation in biological tissue often requires high input energies and hence carries an elevated risk of damage. Introduction of exogenous or artificial cavitation nuclei may substantially reduce the input energy required to produce the broad range of effects discussed above and further introduces additional effects that may not be possible with US alone. Cavitation nuclei include gas bubbles26,44, liquid droplets45,46,47 and solid particles48,49,50, with nanoscale cavitation nuclei being an emergent area of investigation for their benefits in terms of prolonged circulation time, improved extravasation and prolonged cavitation activity49,51,52,53.
The most commonly used nuclei are gas microbubbles (MBs), originally used as contrast agents in diagnostic imaging. They are typically 1-2 micrometers in diameter and contain a core of a high-molecular-weight gas with low aqueous solubility in the surrounding medium. The core is surrounded by a protective lipid, protein, or polymer shell most commonly consisting of&#160;phospholipids54. When exposed to a US field, the compressibility of the MBs causes them to undergo volumetric oscillations, consequently producing&#160;strong acoustic scattering, which is responsible for the success of MBs as a contrast agent. As mentioned, these oscillations also lead to the aforementioned mechanical, thermal, and chemical effects that can be harnessed in therapeutic applications. The MB coating process also offers a mechanism for encapsulating drugs within the MB structure and for attaching drugs and/or targeting species to the MB surface. This technique facilitates the triggered release of drugs to reduce systemic toxicity55. It has also recently been shown that material from the MB surface may be transferred to biological structures, enhancing drug delivery through so called &#8220;sonoprinting&#8221;56,57,58.
Monitoring of US-mediated cavitation activity can provide insights into the resultant biological effects both in vitro and in vivo and potentially allows for the tuning and optimization of these effects. The two most widely applied methods for monitoring cavitation activity are i) optical, which use ultra high speed video microscopy and are&#160;generally not feasible in vivo; and ii) acoustic, which record&#160;the re-radiated sound fields produced by oscillating and/or collapsing bubbles. Both the amplitude and frequency components of the acoustic signal contain information on bubble behavior. Low concentrations of bubbles at low incident US amplitudes have been shown to produce predominantly harmonic emissions (integer multiples of the driving frequency)59. As driving pressures increase, the bubble emission spectrum may also contain fractional components known as subharmonics and ultraharmonics60 that indicate stronger nonlinear behavior, as well as broadband noise, which is&#160;indicative of inertial cavitation. Integer harmonics are a primary indicator&#160;of bubble oscillation but can also be caused by non-linearities anywhere in an experimental system, e.g., due to non-linear propagation. By contrast, fractional harmonics and broadband noise are very strongly correlated with bubble dynamics.
The relationship between bubble behavior and the detected acoustic emissions may be complicated by factors including the incident US field, the nucleation environment, and the characteristics of the detection pathway60. Nevertheless, important information about bubble behavior and their interactions with cells can be gained by discerning trends in frequency and energy in the acoustic spectrum. These data can also provide valuable information that can be used to form the basis for clinical treatment monitoring techniques. To fully exploit this information, the development of robust, translatable, and reproducible experimental methods is required.
Currently there is substantial variation in reported protocols for designing systems and conducting studies to support development of cavitation-assisted therapies. In terms of the apparatus, a range of design approaches has been undertaken. Several groups have made use of parallel-plate chambers56,61,62,63, either custom built or commercially available (e.g., OptiCell, ThermoFisher Scientific). Hu et al.&#160;(2013) developed a cell chamber coupled with an US sonication module and real-time confocal imaging64, Carugo et al. (2015) used a system comprising a commercially available cell culture dish with a custom-made PDMS lid to allow for submersion in a water bath during US exposure65, and Pereno et al. (2018) used a device consisting of layered acoustofluidic resonators that allow for simultaneous optical and acoustic characterization of bubble dynamics and bubble-cell interactions66. The use of custom-fabricated and application-specific designs complicates characterization of the US field and other environmental exposure conditions, making cross study comparisons challenging. For example, there is considerable variation in the US parameters identified for achieving successful sonoporation, which include center frequencies ranging from 0.02 to 15 MHz, duty cycles varying from 1% to continuous wave, and rarefactional pressures ranging from 0.1 to 20 MPa23,64,67,68,69,70 (Table 1). There is similarly considerable variation in the spectral components (harmonics, sub-harmonics etc.) that have been identified as being associated with particular bioeffects.
The aim of this work, therefore, is to provide an easily reproducible system design and implementation framework for the in vitro study of cavitation-induced cellular bioeffects with the specific inclusion of a cavitation monitoring capability.

<table>
	<tbody>
		<tr>
			<td>Absorber</td>
			<td>Precision Acoustics</td>
			<td>APTFlex F28 panel</td>
			<td>1.0 cm standard thickness</td>
		</tr>
		<tr>
			<td>Amplifier (power)</td>
			<td>E&#38;I Ltd.</td>
			<td>1040L</td>
			<td>400W power amplifier to drive ultrasound source</td>
		</tr>
		<tr>
			<td>Amplifier (pre)</td>
			<td>Stanford Research Systems</td>
			<td>SR445A</td>
			<td>Fixed gain multi-stage preamplifier for PCD signals</td>
		</tr>
		<tr>
			<td>Aquarium heater</td>
			<td>Aquael</td>
			<td>Ultra 50W</td>
			<td>Different models for different tank sizes.</td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>TiePie Engineering</td>
			<td>HS5-110-XM</td>
			<td>Extended memory option: 32M points per channel</td>
		</tr>
		<tr>
			<td>Hydrophone</td>
			<td>Precision Acoustics</td>
			<td>FOH</td>
			<td>0.01 mm diameter sensitive area minimises directivity effects</td>
		</tr>
		<tr>
			<td>Microbubbles</td>
			<td>Bracco</td>
			<td>SonoVue</td>
			<td>FDA approved microbubbles</td>
		</tr>
		<tr>
			<td>PCD mirror (SAT3)</td>
			<td>Olympus NDT</td>
			<td>F-102</td>
			<td>90 degree beam reflection</td>
		</tr>
		<tr>
			<td>PCD transducer</td>
			<td>Olympus NDT</td>
			<td>V320-SU</td>
			<td>Immersion transducer, 7.5MHz</td>
		</tr>
		<tr>
			<td>PCD waterproof cable</td>
			<td>Olympus NDT</td>
			<td>BCU-58-1 W</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS (SAT2 compartment lid)</td>
			<td>Corning</td>
			<td>Sylgard 184</td>
			<td>See Carugo et al. (2015) for preparation guidelines</td>
		</tr>
		<tr>
			<td>Polymer rod (SAT2 seal)</td>
			<td>Zeus</td>
			<td>PTFE monofilament</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber plug (SAT3 lid/seal)</td>
			<td>VWR</td>
			<td>391-2101</td>
			<td>6mm bottom dia., 8mm top dia., red</td>
		</tr>
		<tr>
			<td>Signal generator</td>
			<td>Agilent</td>
			<td>33250</td>
			<td>Waveform generator for ultrasound source</td>
		</tr>
		<tr>
			<td>Substrate for cell exposure compartment, SAT2</td>
			<td>Ibidi</td>
			<td>&#181;-Dish 35mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate for cell exposure compartment, SAT3</td>
			<td>Corning</td>
			<td>Transwell 6.5mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound source (SAT3)</td>
			<td>Sonic Concepts</td>
			<td>H107 with central hole</td>
			<td>Use of a HIFU-capable source allows pressures &#62;1MPa to be generated both at the focus and pre-focally for expanded spatial coverage</td>
		</tr>
	</tbody>
</table>

studying cavitation enhanced therapy

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to Alzheimer&#8217;s disease. Cavitation - the formation and subsequent motion of bubbles within an ultrasound field - represents a key phenomenon underpinning many of these treatments. There remains, however, considerable uncertainty regarding the detailed mechanisms of action by which cavitation promotes therapeutic effects and there is a need to develop reliable monitoring techniques that can be implemented clinically. In particular, there is significant variation between studies in the exposure parameters reported as successfully delivering therapeutic effects and the corresponding acoustic emissions. The aim of this paper is to provide design guidelines and an experimental protocol using widely available components for performing studies of cavitation-mediated bioeffects, and&#160;include real-time acoustic monitoring. It is hoped that the protocol will enable more widespread incorporation of acoustic monitoring into therapeutic ultrasound experiments and facilitate easier comparison across studies of exposure conditions and their correlation to relevant bio-effects.

Figure 4 shows examples of time and frequency domain PCD responses, illustrating three distinct cavitation behaviors. All data were collected on SAT3 using SonoVue MBs diluted 5x in PBS, with a final concentration of ~2*107 MBs/ml. The temperature for all examples in this section was 19 &#177; 1 &#176;C. The US source was driven with a 2.0 ms pulse at 0.5 MHz to achieve incident peak negative pressures of 0.20 (Figure 4A and 4B), 0.30 (Figure 4C and 4D), and 0.70 MPa (Figure 4E and 4F). The signal recordings began 1.4 ms before the t = 0 start of the US pulse. The inset traces show the signal as recorded (red) and with a 2 MHz high pass filter (blue) for a time window centered at the time of flight from source to cell exposure compartment to PCD. The low-level response before this time is due to directly received radiation from the source, which is common in configurations where the PCD is behind the US source.
At the lowest incident pressure, the PCD response consists entirely of integer harmonics of the 0.5 MHz fundamental US frequency. Increasing from 0.20 to 0.30 MPa results in pronounced ultraharmonics in the spectrum in addition to further elevated integer harmonics. The time domain waveforms at these two pressures look similar, although the 0.30 MPa results show more variability over the pulse duration. At the highest pressure, the time domain waveform amplitude has grown nonlinearly relative to the lower pressures as a result of clearly elevated broadband noise visible in the spectrum. This noise is commonly considered to be a result of inertial cavitation and in this example, corresponds to destruction of MBs.
To see this more clearly, PCD responses as a function of time are shown in Figure 5. In the left panel (Figure 5A), full spectra are shown over a 50 second exposure time, during which the source emitted 2.0 ms pulses every 0.20 seconds. Corresponding total, harmonic and broadband powers are shown in the right panel (Figure 5B). The US was turned on at t =3 .0 s, at which time large-amplitude broadband responses were seen. The initial spike is thought to correspond to the destruction of the largest bubbles in the suspension (SonoVue is polydisperse) and is a common observation in cavitation experiments with shelled bubbles and even with non-degassed media (e.g., PBS).
After a few seconds, the broadband response rapidly diminished, apparently due to bubble destruction, and the signal is predominantly comprised of harmonics. This suggests that the freed gas and remaining MBs are vibrating stably and non-inertially. At t~50s, the broadband component has fallen to the level of the original background noise. Exposure tests like this are therefore important when trying to understand the timescales during which different bubble effects may be acting upon the cells in the chamber.
Bubbles are likely to translate in response to radiation forces generated during US exposure and movement of MBs in and out of the PCD field of view can lead to increased variability in the monitored cavitation signal, especially when dealing with dilute suspensions. The sensitive region of the PCD should therefore span as much of the cell exposure surface as possible. A comparison of the&#160;responses&#160;focused and unfocused PCDs with identical center frequencies (see Figure 2) is shown in Figure 6, using a 20:1 dilution of MBs in normal PBS is SAT2. The time and sample-averaged spectra in panel Figure 6A&#160;show that the unfocused PCD contains a stronger broadband response, accompanied by reduced sample-to-sample variability in both harmonic (Figure 6B) and ultraharmonic powers (Figure 6C).
It is important to recognize that media used for in vitro cell work are not degassed and may present an enhanced background level of bubble activity. Figure 7 shows the response in SAT2 of PBS used in its supplier-provided form and after two hours of degassing under vacuum, after which the air saturation was reduced from 92% to 46% as determined with an optical sensor (PreSens, Germany). The spectra in Figure 7A were averaged over exposure time and repeats with five independent samples, and clearly show show clearly elevated ultraharmonics in normal PBS. Powers summed over three harmonics (Figure 7B) are well within the standard deviation of each experimental output. By contrast, the ultraharmonic sums in Figure 7C show that normal PBS has nearly an order of magnitude higher level and substantially higher variability between samples. These examples indicate that a common cell-compatible medium may exhibit behaviors that could be (incorrectly) attributed to the presence of MBs. Since it is usually impractical to degas culture medium due to the negative impact upon cells and/or cavitation agent stability, it is critical to perform suitable controls in any cavitation-related study.
<img alt="Figure 1" src="/files/ftp_upload/61989/61989fig1.jpg" /> 
Figure 1: Illustrations of two US exposure system designs incorporating cavitation monitoring: SAT3 (D-F).&#160;(A) SAT2 annotated assembly with side wall removed for clarity. (B) SAT2 with side wall intact. (C) SAT2 cell exposure compartment, disassembled. (D) SAT3 annotated assembly. (E) SAT3 in normal (left) and lensed (right) configurations for beam width matching at different frequencies. (F) SAT3 cell exposure compartment, disassembled. <a href="https://www.jove.com/files/ftp_upload/61989/61989fig1large.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/61989/61989fig02.jpg" /> 
Figure 2: Calculations of half amplitude pressure field contours for 12.7 mm diameter unfocused (left) and spherically focused (right) transducers.&#160;Frequencies of 2, 4 and 8 MHz are shown as red, blue, and green contours, respectively, for a PCD element at the coordinate origin (0,0). The outermost contours of the unfocused device are relatively insensitive to frequency, but the interior structure is frequency dependent. The spherically focused field contracts as frequency increases, but inside the contours, the fields vary smoothly. <a href="https://www.jove.com/files/ftp_upload/61989/61989fig02large.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/61989/61989fig03.jpg" /> 
Figure 3: Instrumentation for cavitation signal conditioning and recording (blue arrows), US source excitation (red lines), and data acquisition triggering. <a href="https://www.jove.com/files/ftp_upload/61989/61989fig03large.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/61989/61989fig04.jpg" /> 
Figure 4: Time (left) and frequency (right) domain PCD responses recorded with MBs diluted 5x in PBS.&#160;Incident peak negative pressures were (A, B) 0.2 MPa, (C, D) 0.4 MPa, (E, F) 0.7 MPa, all at 0.5 MHz. Signal recordings begin 1.4 ms before the t=0 start of the 2.0 ms duration ultrasound pulse. (A, C, E) Time domain signals (red) are shown on a fixed vertical scale, indicating how the response level changes with incident pressure. The inset traces show the signal as recorded (red) and with a 2 MHz high pass filter (blue) for a time window centered at the time of flight from source to cell exposure compartment to PCD. (B, D, F) Noise and signal power spectral densities are calculated for t&#60;0 and t&#62;0, respectively. <a href="https://www.jove.com/files/ftp_upload/61989/61989fig04large.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/61989/61989fig05.jpg" /> 
Figure 5: Spectrum histories over a 50 second exposure of a suspension of MBs&#160;diluted 5x in PBS.&#160;(A) Full spectra and (B) total, harmonic and broadband signal powers, all as a function of time. Drive conditions were 0.5 MHz, 0.7 MPa peak negative pressure, 2.0 ms pulse duration, 200 ms pulse repetition period. <a href="https://www.jove.com/files/ftp_upload/61989/61989fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61989/61989fig06.jpg" /> 
Figure 6: Effect of PCD focusing geometry recorded with a 20:1 dilution of microbubbles in normal PBS.&#160;Drive conditions were: 1.0 MHz, 0.50 MPa peak negative pressure, 3.0 ms pulse duration, 10 ms pulse repetition period. (A) Full spectra averaged over exposure time and three independent sample repeats. (B) Power in 3, 4 and 5 MHz harmonics, and (C) power in 2.5, 3.5 and 4.5 MHz ultraharmonics. Thick lines are sample means, shaded areas indicate +/- 1 standard deviation. <a href="https://www.jove.com/files/ftp_upload/61989/61989fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61989/61989fig07.jpg" /> 
Figure 7: Effect of degassed media recorded with PBS.&#160;(A) Full spectra averaged over exposure time and five independent sample repeats. (B) Power in 3, 4 and 5 MHz harmonics, and (C) power in 2.5, 3.5 and 4.5 MHz ultraharmonics. Thick lines are sample means, shaded areas indicate +/- 1 standard deviation. Drive conditions were 1.0 MHz, 0.50 MPa peak negative pressure, 1.0 ms pulse duration, 200 ms pulse repetition period. <a href="https://www.jove.com/files/ftp_upload/61989/61989fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Unit</td>
			<td>Minimum</td>
			<td>Maximum</td>
		</tr>
		<tr>
			<td>frequency</td>
			<td>MHz</td>
			<td>0.02</td>
			<td>15</td>
		</tr>
		<tr>
			<td>pressure (peak negative)</td>
			<td>MPa</td>
			<td>0.1</td>
			<td>20</td>
		</tr>
		<tr>
			<td>pulse length</td>
			<td>cycles</td>
			<td>1</td>
			<td>CW</td>
		</tr>
		<tr>
			<td>duty cycle</td>
			<td>%</td>
			<td>1</td>
			<td>CW</td>
		</tr>
		<tr>
			<td>exposure time</td>
			<td>s</td>
			<td>10</td>
			<td>1000</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of the range of reported parameters facilitating sonoporation in vitro.

Watch this Scientific Journal Video about Studying Cavitation Enhanced Therapy at JoVE.com

Studying Cavitation Enhanced Therapy

The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to ...

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5.1K Views.  University of Oxford. The presented experimental protocol can be used to perform real time measurements of cavitation activity in a cell culture device with the aim of enabling investigation of the conditions required for successful drug delivery and/or other bioeffects.

Video: Studying Cavitation Enhanced Therapy

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett&#8217;s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett&#8217;s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&amp;#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.