IN DEGREE PROJECT MEDICAL ENGINEERING,SECOND CYCLE, 30 CREDITS

, STOCKHOLM SWEDEN 2019

Acoustic Droplet Vaporization of Perfluorocarbon Filled Microdroplets

Akustisk evaporation av mikrodroppar fyllda med perfluorokarbon

DIDRIK NIMANDER

KTH ROYAL INSTITUTE OF TECHNOLOGYSCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

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Acknowledgements

I would like to express my gratitude to Dmitry Grishenkov and Morteza Ghorbani for their guidance,

their valuable and constructive input throughout the project, and for their willingness to give their

time.

I would also like to thank Hongjian Chen and Ksenia Loskutova for their help with lab equipment

and procedures, as well as Peter Arfert for his help with 3D-printing the holder used and Martin

Viklund for reviewing my work.

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Abstract

The use of perfluorocarbon filled droplets for use as Phase Changing Contrast Agents (PCCAs) is

a promosing field. These capsules also have potential to be used for mediated drug delivery. The

phase change, which has given the capsules their name, is the process when the capsule transforms

from a droplet into a bubble. This process is referred to as Acoustic Droplet Vaporization (ADV)

and can be induced with the use of ultrasonic waves.

In this study a new type of Perfluorpentane (PFC5) capsules which are stabilized with Cellulose

Nano Fibers (CNF) have been evaluated for its potential as a PCCA. To investigate this potential,

a setup was designed in which the capsules could be exposed to ultrasound waves. Following the

ultrasound exposure the capsules were visualized under a light transmission microscope.

The experiments were conducted for different combinations of ultrasound parameters. For each

combination eight volume distributions were created, in which two of them as reference cases were

not exposed to ultrasound waves. Six cases with the ultrasound firing with different levels of acoustic

power, resulting in peak negative pressures ranging from 0.144 to 0.291 MPa. The results showed

that ADV could be observed when the frequency of the acoustic wave is 3.5 MHz, the pulse repetition

frequency is 500 and the burst width is set to 12 cycles. The Peak Negative Pressure (PNP)-threshold

for ADV is about 0.200 MPa. When the burst width is set to 8, ADV is also observed however,

to a lesser extent then when it is set to 12. These results indicate that the CNF-stabilized PFC5

capsules are promising droplets with a potential future as an alternative to currently used PCCAs.

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Abbreviations

MB - Microbubble

AD - Acoustic Droplets

ADV - Acoustic Droplet Vaporization

PFC - Perfluorocarbon

PFC5- Perfluoropentane

PCCA - Phase-changing Contrast Agent

IC - Inertial Cavitation

PRF - Pulse Repetition Frequency

PNP - Peak Negative Pressure

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Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2 Preparation of CNF-stabilized PFC5 droplets . . . . . . . . . . . . . . . . . . . . . . 3

2.3 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.4 Experimental protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.4.1 Parameter changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.5 Post-processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.5.1 ImageJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.5.2 Distribution plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.6 Conversion of electrical output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.1 Results with a burst width of 12 cycles and a PRF of 500 . . . . . . . . . . . . . . . 7

3.2 Results with a burst width of 8 cycles and a PRF of 500 . . . . . . . . . . . . . . . . 10

3.3 Results with a burst width of 4 cycles and a PRF of 500 . . . . . . . . . . . . . . . . 10

3.4 Results with a burst width of 12 cycles and a PRF of 100 . . . . . . . . . . . . . . . 10

4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

4.1 Parameter choices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.2 Omission of larger diameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.3 Effects of setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4.4 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Appendicesdddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddddllll

Appendix A State of the art reportdddddddddddddddddd dll

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

A State of the art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

A.1 Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

A.1.1 Properties of the ultrasound wave . . . . . . . . . . . . . . . . . . . . . . . . 1

A.1.1.1 Frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

A.1.1.2 Acoustic Impedance . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

A.1.1.3 Attenuation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

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A.1.1.4 Thermal and Mechanical index . . . . . . . . . . . . . . . . . . . . . 2

A.1.1.5 Burst width . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

A.1.1.6 Repetition rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

A.1.2 Transducer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

A.1.2.1 Single crystal transducer . . . . . . . . . . . . . . . . . . . . . . . . 3

A.2 Capsules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

A.2.1 Microbubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

A.2.2 Acoustic droplets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

A.2.2.1 Manufacturing process of acoustic droplets . . . . . . . . . . . . . . 5

A.3 Acoustic Droplet Vaporization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

A.3.1 Properties affecting ADV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

A.3.2 Effects of constrained geometry . . . . . . . . . . . . . . . . . . . . . . . . . . 6

A.4 Potential areas of use for ADV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

A.4.1 Contrast agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

A.4.2 Drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

A.4.3 HIFU treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

A.4.4 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

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1. Introduction

Within clinical ultrasound, Microbubbles (MB) are commonly utilized as a contrast agent [1]. A

new promising application of microbubbles in combination with ultrasound is what is known as

ultrasound controlled mediated drug delivery, which is a technique to utilize the small bubbles to

deliver for example chemotherapeutic drugs directly to tumours [2].

There are however, a number of disadvantages related to the use of MBs in vivo. One such dis-

advantage is that MBs often have a short lifetime after injection [3]. The use of sub-micron liquid

perfluorocarbon (PFC) filled capsules is therefore of interest for the related communities, the cap-

sules are referred to as Phase Changing Contrast Agent (PCCA) as well. The PCCAs, which are

considered as acoustic droplets (ADs). The capsules are filled with a liquid and are encapsulated

within a shell. The shell is commonly made up of lipids or polymers. The PCCA can then be con-

verted from a droplet into a microbubble by forcing the liquid inside to go through a phase change

into gas. This is done with the help of ultrasound and the process is known as Acoustic Droplet

Vaporization (ADV) [3]. When the droplets undergo the transformation into bubbles, they grow in

size with a factor of approximately 4 to 6 [4].

One advantage of using PCCAs as a contrast agent, is that when the capsules are in the AD-state

they have an increased half-time in vivo compared to traditional MB’s. Another advantage is the

smaller size of the droplets compared to the bubbles [3]. Within drug delivery, most drugs that are

used are in liquid form. Mixing liquids is easier and more practical than mixing a liquid with a

gas, therefore PCCAs are considered as a strong alternative within drug delivery. Phase changing

capsules in tandem with ADV have a number of promising areas of use. Most of them are shared

with current microbubbles. The areas of use are discussed further in section A.4 of Appendix A.

This study concerns a new type of PFC5 capsules which are reinforced by Cellulose Nano Fibers

(CNF). The aim is to evaluate the behavior of these capsules, when they are exposed to ultrasound

waves under different conditions, such as altering the burst widt of the ultrasound wave. Thus, it is

possible to determine their potential for use as a PCCA through investigation of the ADV process.

These bubbles are very stable and non-toxic implying that they could be a promising alternative to

currently used capsules [5].

To do so, the Peak Negative Pressure (PNP) which is required to induce the PFC5 droplets is

investigated with the use of a single crystal transducer (see section A.1.2.1). A PFC5 droplet

suspension will be subjected to ultrasound waves with varying acoustic parameters. Following the

ultrasound exposure, the suspension is visualized under a microscope (ECLIPSE Ci-S, Nikon, Tokyo,

Japan). The images obtained from the microscope is then analyzed and volume distributions are

generated.

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1.1 Aim

The aim of this thesis is to design an ultrasound set-up, which is used to asses the effect of ultrasound

waves on a new type of PFC5 capsules in a confined geometry. Different ultrasound properties such as

Pulse Repetition Frequency (PRF), burst width and acoustic power are varied in order to investigate

the pressure threshold for ADV. The effect of the ultrasound exposure is visualized and quantified

using a transmission light microscope. The ADV threshold is of great interest when investigating

whether or not this new type of capsule has potential to be used in diagnostic and therapeutic

applications, for example as a contrast agent or for drug delivery.

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2. Materials and Methods

2.1 Materials

Perfluoropentane (PFC5, 99%) was purchased from Apollo Scientific (City, U.K.). Bleached sulfite

pulp (from Nordic Paper Seffle AB, Sweden) was used in the production of the cationic cellulose

nanofibers (CNFs). The CNF suspension (1.32 wt%) were prepared as described previously [5]. The

amount of cationic groups, obtained by conductometric titration, was 0.13 mmol per g fiber [6].

2.2 Preparation of CNF-stabilized PFC5 droplets

A suspension of CNF (0.28 wt%) was prepared by diluting the stock CNF with MilliQ-water (pH

of diluted CNF suspension was 9.5). The CNFs were dispersed with an ultrasonic liquid processor

(Sonics Vibracell W750, U.S.). The suspension was treated at an amplitude of 90% for 180 s (using

a ” tip) as described previously [5]. The CNFs were ca. 4 nm in width and with a length in the

micrometer range. The suspension was brought to room temperature and afterwards 36 g of the (0.28

wt%) CNF suspension was mixed with 1 g of PFC5. The mixture was then processed for another

60s at an amplitude of 80% under ice-cooling to obtain the stock suspension of CNF-stabilized PFC5

droplets.

The suspension was diluted to achieve a reasonable concentration of capsules for imaging under a

microscope. 1000 ml of PFC5 suspension was diluted with 9000 ml MilliQ water to achieve a 1:9

ratio. The same sample of stock PFC5 suspension was used for each experiment and a new dilution

was created for each session in the lab.

2.3 Experimental setup

The setup went through a number of designs before a final version was settled upon. The setup

consisted of a water bath with a flat transducer (V382-SU Olympus NDT, Waltham, MA) integrated

into the bottom of the tank, a plastic tube with inner diameter of 500 µ m(Zeus Inc., Orangeburg,

SC, USA), a holder and a syringe (BD Plastipak 1 ml, Becton Dickinson, Franklin lakes, NJ, USA).

The transducer was powered by a Controlled Ultrasonic System ( SNAP Mark IV, Ritec, Inc.,

Warwick, RI, USA) as shown in Figure 2.1. The slide was inserted into the microscope equipped

with a 10x objective after the suspension had been dropped onto it from the end of the tube.

The holder depicted in Figure 2.2, was designed and drawn up in Solide Edge ST10 (Siemens,

Munchen, Germany). The drawing was then sent to a workshop, where it was 3D-printed. Weights

were later added to keep the holder from movement when submerged under water.

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Figure 2.1: Schematic of the experimental setup. Figure 2.2: CAD-Drawing ofthe holder.

2.4 Experimental protocol

When a final setup was settled upon, the experiments could be initiated. The setup was used to

expose the suspension to the ultrasound. A number of acoustic parameters were changed in between

runs in an attempt to characterize and optimise the settings for the ADV of the capsules. All

experiments were performed in the same way. First all parts of the setup were prepared and made

ready. The plastic tubing was inserted into the holder, which was then submerged into the water

bath and placed correctly over the transducer. The PFC5-suspension was gently mixed by hand

and then the syringe was used to draw up the suspension. The syringe was then taped to the side

of the water bath and connected to the plastic tube via a needle, which the tube was glued on to.

The ultrasound was activated and the syringe was then used to propel the suspension through the

tube and through the holder over the transducer. The other end of the tube was then placed over a

microscopy slide (VWR International, Radnor, PA, USA) and the suspension was allowed to drop

from the end of the tube onto the slide. Five drops were added to the slide before the slide was

placed under the microscope and images of the capsules within these drops were obtained.

The procedure was repeated 8 times in total for each experiment, in which the first one was without

ultrasound for reference. With the ultrasound activated the attenuation of the electrical output was

set to 10 dB and then decreased to 0 dB in intervals of 2 dB. Finally the second batch of reference

images were taken after the experiments.

The tube was cleaned between each repetition. The cleaning was performed by flushing the tube

with EcoSolv A, 99.5% (Solveco chemicals AB, Rosersberg, Sweden) ethanol solution, which was left

inside the tubing for a couple of minutes. Following this, the tubing was flushed with MilliQ-water

and finally flushed out with air to empty the tubing before the next repetition.

2.4.1 Parameter changes

This experiment was repeated four times. The frequency was set to 3.5 MHz for each repetition.

PRF and burst width were varied separately between each experiment. The parameter combinations

used for each experiment is presented in table 2.1.

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Table 2.1: Combinations of acoustic parameters for a frequency of 3.5 MHz.

Experiment PRF(Pulses per second) Burst width(Number of cycles per pulse)1 500 122 500 43 500 84 100 12

2.5 Post-processing

The post-processing involved the processing of the raw images in ImageJ (version 1.8.0 172, National

institutes of health, USA) and analyzing them with the aid of an inhouse MATLABTM (MathWorks

Inc., Natick, MA, USA) script to achieve normalized distributions.

2.5.1 ImageJ

Firstly the images had to be processed so to obtain the size distribution of the capsules using ImageJ.

The software has two built in functions which were used to obtain the size data of the particles.

The images were made binary, and then analyzed using the function ”Analyze particles”. The size

limitation parameter was set to ”2-infinity” and the circularity constraints to ”0.3-1”. There were

many non-capsules due to a lot of dust and other ”artefacts” which could be interpreted as particles

by the software. Therefore non-capsule particles, such as dust, were erased after the image had been

made binary. The different steps of the image being processed are shown in Figure 2.3, 2.4 and 2.5.

Figure 2.3: Originalimage of the capsule sus-

pension.

Figure 2.4: The sameimage made binary.

Figure 2.5: The imageafter manual clean-up.

2.5.2 Distribution plots

The data obtained from ImageJ was inserted into an Excel workbook. The MATLAB script was

used to generate the data to create a volume distribution with a fitted curve for each subset of

the data. The mean and standard deviation of both the diameter and volume of the particles were

calculated from the data used in the script. For all created plots a cut-off for diameters larger than

35 was implemented.

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2.6 Conversion of electrical output

As previously mentioned the experiments were conducted using a range from 10 to 0 dB attenuation

of the electrical output from the generator. To make the results viable, the PNP for the different

levels of attenuation was needed. The data from a hydrophone test of the transducer used was

available, and it is presented in Figure 2.6.

Figure 2.6: Plot showing the relationship between electrical output from the ultrasound generatorand PNP of the resulting acoustic wave [7].

This graph shows how the PNP depends on the attenuation of the electrical output all the way up

to 20 dB attenuation. The relevant data points have been lifted out and inserted into Table 2.2. All

values have been rounded to 3 decimal places.

Table 2.2: Relevant conversion data points.

dB 0 2 4 6 8 10PNP (MPa) 0.291 0.260 0.228 0.200 0.169 0.144

All results were presented using the PNP values from Table 2.1 instead of the settings of the elec-

trical attenuators applied.

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3. Results

The data obtained from experiments have been turned into volume distribution plots. Two statistical

values which are important to interpret the resulting data was then plotted from the same dataset

as explained in chapter 2. For all combinations of acoustic parameters used, the frequency was 3.5

MHz. The combination with the most evident occurrence of ADV was when the burst width was

set to 12 cycles and the PRF was set to 500.

3.1 Results with a burst width of 12 cycles and a PRF of 500

In figure 3.1 the volume distributions are presented. Figures 3.1 A and B show the volume distri-

bution of the reference measurements, in which the suspension was not exposed to any ultrasound

pressure. The figures both show that the majority of the distribution is grouped around a peak that

is located at approximately 8 µm. The Gaussian is centred around this peak, and there is only a

small normalized frequency distributed at diameter values above 15 µm.

In the next Figure, 3.1 C the sample has been exposed to ultrasound with a PNP of 0.144 MPa.

There is no major change in the distribution, however the normalized frequency of capsules with a

diameter under 15 µm is decreasing. A larger part of the capsule population seems to have shifted

to slightly larger sizes. For figure 3.1 D the PNP has been increased to 0.169 MPa and the small

changes in the distribution is similar to the ones seen in Figure 3.1 C.

The distribution for a PNP value of 0.200 MPa is presented in figure 3.1 E. The volume distribution

was noticeably affected. The normalized frequency of the group of capsules around 10 µm decreased

significantly and the peak which was seen earlier was no longer evident. The distribution shifted

significantly towards larger diameters and the existence of capsules as large as 25 to 30 µm was

observed. Figure 3.1 F, shows the distribution for a PNP of 0.228 MPa. The distribution had an

even larger shift in the distribution compared to the previous cases. Larger quantity of capsules

with a diameter≥ 20 µm was observed as well.

The PNP was increased further to 0.260 MPa, see Figure 3.1 G. The distribution still shows a

shift when compared to the reference distributions. However compared to Figures 3.1 E and F the

distribution remained closer to the reference with less shift in sizes. Figure 3.1 H, with a PNP of

0.291 MPa shows less difference from the reference plots. A large part of the volume distribution

is located at diameters below 15 µm. There are also two extreme values in the distribution which

account for a considerable part of the normalized frequency.

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((a)) Reference with no ultrasound

activated(before)

((b)) Reference with no ultrasound

activated(after).

((c)) Volume distribution with 0.144

MPa PNP.

((d)) Volume distribution with 0.169

MPa PNP.

((e)) Volume distribution with 0.200

MPa PNP.

((f)) Volume distribution with 0.228

MPa PNP.

((g)) Volume distribution with 0.260

MPa PNP.

((h)) Volume distribution with 0.291

MPa PNP.

Figure 3.1: Figures depicting the Volume distribution for PRF=500 and Burst width= 12 forPNP ranging from 0.144 to 0.291 MPa. The diameter of the imaged capsules plotted against the

normalized frequency of volume for the capsules of that diameter.

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The mean and standard deviations were calculated from the raw data which was presented in Figure

3.1. Figure 3.2 shows the ultrasound waves effect on the diameter. The mean values and the standard

deviation are around the same values for all PNPs, while the exception being the values for when

the PNP was 0.200 MPa.

Figure 3.2: Plot of the mean diameter of the capsules for each level of acoustic power for Burstwidth=12 and PRF=500.

In Figure 3.3 the mean value as well as the standard deviation of the volume of capsules are shown.

The mean values are similar for the two reference batches as well as for PNP set to 0.144 and 0.169.

The mean and, especially, the standard deviation increase significantly for PNP≥0.200 MPa. The

increase is the most dramatic for the PNP values of 0.200 and 0.228 MPa. The results presented

in Figure 3.3 indicates two distinct trends before and after the PNP of 0.2 MPa. It is clearly seen

that there is a sharp increase in the volume at the PNP of 0.2 MPa, which is extended to the higher

values of PNP. This increasing trend is in coincidence with the normalized frequency of the volume

distributions illustrated in Figure 3.1 which implies the arrival of ADV and its continuation till the

PNP of 0.291 MPa.

Figure 3.3: Plot of the mean volume of the capsules for each level of acoustic power for burstwidth=12 and PRF=500.

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3.2 Results with a burst width of 8 cycles and a PRF of 500

The burst width was reduced to 8 cycles while the PRF was still 500. In appendix B, the Figure

B1 depicts the volume distributions for all settings of PNP. The images show a similar trend to the

one observed in Figure 3.1. For PNP values ≤ 0.169 MPa, the effect on the volume distribution is

minimal, as shown in Figure B.1 A-D. The bulk of the normalized frequency around 10 µm seen in

Figure 3.1 is also seen in this case. When PNP values reach higher values, see Figure B.1 E-H, a

shift in the volume distribution can be seen. The shift is not as evident as for the corresponding

sub-figures in Figure 3.1. The reduction of the bulk of normalized frequency around diameters of 10

µm is not as pronounced as observed in Figure 3.1 but still present.

For these parameter settings, there is no significant change in the mean values across the plot. The

standard deviation shows a similar behaviour as when the burst width was set to 12 cycles, but

the effect is less exaggerated. The significant increase in standard deviation occurs when the PNP

reaches 0.200 MPa, as shown in Figure 3.5. The trends described for Figure 3.3 is present in Figure

3.4 as well, coinciding with the normalized frequency of the volume distribution as well, see Figure

B.1. Figure 3.4 shows that for the mean diameter, the standard deviation is barely affected.

Figure 3.4: Plot of the mean diameter ofthe capsules for each level of acoustic power

for burst width=8 and PRF=500.

Figure 3.5: Plot of the mean volume ofthe capsules for each level of acoustic power

for burst width=8 and PRF=500.

3.3 Results with a burst width of 4 cycles and a PRF of 500

The Burst width was then decreased to 4 cycles, while all other acoustic parameters were kept

constant. The distributions can be seen in Figure B.2. The plots for mean, and standard deviation

are depicted in Figure C.5 and C.6 and there is no significant change in mean or standard deviation

for any PNP value. The volume distributions, see Figure B.2, only have minor changes in their

appearance. For a PNP of 0.228 and 0.291 MPa there are a small number of capsules with diameter

above 20 µm.

3.4 Results with a burst width of 12 cycles and a PRF of 100

The burst width was set to 12 cycles in the next experiment in which the PRF was investigated.

The PRF was decreased too 100 pulses per second. For this combination of acoustic parameters,

the results were similar to those where the burst width was set to 4 cycles. There appears to be no

significant impact from the ultrasound exposure for any of the tested PNP values. Figure B.3 shows

the volume distributions from these tests while Figures C.7 and C.8 show the mean plots.

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In Figures 3.6 and 3.7, two microscopy images were included to show some size difference of the

capsules visually. The smaller capsules in Figure 3.6 are substantially smaller than the largest one

illustrated in Figure 3.7, which has a diameter of approximately 30 µm.

Figure 3.6: One of the reference images ofthe suspension for the experiments with aPRF of 500 and burst width=12 was used.

Figure 3.7: One of the images of the sus-pension for the experiments with a PRF of500 and burst width=12 with PNP= 0.291

was used.

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4. Discussion

The results of the initial set of acoustic parameters show that at 0.200 MPa there is a relatively

large change in mean value of the volume of the capsules. There is also a substantial increase of

the standard deviation for the volume of the capsules. This suggests that ADV has occurred, since

when the capsules undergo a phase change they also grow in size. The increase in mean value and

standard deviation, which is seen in Figure 3.3 point to the existence of larger bubbles than before.

The distribution is shifting towards larger sizes, Figure 3.1 E is a good example to show this. It is

evident that the frequency of smaller capsules decreases while it increases for larger capsules. The

reason for the standard deviation being affected a lot while the mean values are not, is that the

large bubbles generated by ADV are few in number. The effect is even bigger in the volume-related

plots. This is due to that the conversion from diameter to volume increases all values by three

orders of magnitude. This exaggerates the difference between the smallest and largest bubbles, in

turn increasing the standard deviation.

The fact that the mean and standard deviation decrease again at higher PNPs can be due to the

increase in power leading to the destruction of some of the capsules, which grow too violently. The

higher values of PRF make the capsules oscillate rapidly and they eventually rupture with increasing

pressure.

Clear signs of ADV was observed with constant PRF and decreased burst width to 8. The results

are similar to the results for 12 cycles. It is seen Figure 3.5, that the volume results show the same

trend regarding standard deviation increase. It is however, not as pronounced as in Figure 3.3. The

mean values do show a slight increase when reaching 0.200 MPa, all though the increase is no larger

than between the two reference batches. The relatively large increase in standard deviation indicates

that, the same as for a burst width of 12 cycles, the ADV threshold lies at 0.200 MPa.

There is another phenomenon for both 8 and 12 cycles which points toward that the capsules have

changed phase and grown from droplets into bubbles, see Figure 3.1. In the reference images and

at lower acoustic power (Figures 3.7 A to D), we see that a majority of the normalized frequency

is located around a peak at approximately 8 to 10 µm. This peak and the surrounding frequencies

decrease for PNP values larger than 0.200 MPa (Figure 3.7 E to H). Combining this with the fact

that larger bubbles of 20 to 30 µm appear at the same time, is evidence pointing toward that the

smaller droplets have undergone a transformation into larger bubbles. This reduction around the

peak and the existence of larger bubbles are present. But they are not as large for 8 cycles, see

Figure B.1. This evidence strengthens the claim that ADV occurs at the same PNP values for both

settings of the burst width, with the effect being larger, when the number of cycles is higher.

It is also worth to mention, that in the volume distribution plots (for instance Figure 3.1) the diam-

eters have been rounded to the nearest 0.5 µm and grouped accordingly. While for the mean plots

(Figure 3.2 and 3.3), this grouping has not been performed.

12

4.1 Parameter choices

For the first experiment, high values were chosen for all acoustic parameters. This in order to

maximize the probability of ADV occurring. The initial experiment indicated that ADV could be

achieved, with a threshold of around 0.2 MPa for these high settings. Therefore, it was decided to

investigate the effect of lowering both PRF and burst width. The burst width was decreased to 4

cycles. The results showed that the PNP required to initiate ADV is higher than the one achieved

with the current setup. Therefore the burst width was chosen mid-way between the first and second

experiments, namely 8 cycles. The next acoustic parameter which was examined to be tested was

the PRF, which was lowered to 100 pulses per cycle. Due to no significant effect for the achievable

PNP values, it was decided to not try and lower it further.

4.2 Omission of larger diameters

In the results, only capsules with a diameter ≥ 35 µm were included when creating the plots. The

reason for this is that; for some experiments, the volume distribution was not affected in a significant

way. However there was a presence of bubbles which were uncharacteristically large. Figure 4.1

shows one of the largest ”normal” bubbles, while Figure 4.2 shows one of the uncharacteristically

large bubbles. The very large bubbles ranged in size quite a lot.

Figure 4.1: Image of a large bubble whichhas gone through a phase transition, a large

”normal” bubble.

Figure 4.2: Image of one of the uncharac-teristically large bubbles.

The majority of these bubbles are a lot bigger than the limit, which is included in the results. The

results therefore do not reflect their existence. This is partly because it is uncertain what lies behind

these bubbles and partly because the volume distribution would only show one very large peak at

the very large bubble if it was included. This causes any information on how the distribution of the

smaller capsules is to be ”lost” in the plot. These very large bubbles could exist just by chance,

they might be a product of ADV or they might be created by a number of capsules coalescing to

form one big bubble.

13

4.3 Effects of setup

After some tests it was discovered that the suspension seemed to be affected by the travel through

the tube. It was clear that a lot of larger droplets seemed to have been lost, when comparing a

suspension, which had travelled through the tubing to one that had not. This effect could be seen

even without the ultrasound activated. The most likely cause is shear forces in the plastic tubing.

Larger capsules travelling close to the edges of the tube can be subjected to different velocities at

the edge compared to the center of the capsule, due to a difference in velocity within the tube. This

gives rise to the shear forces on the capsule, which in turn can cause the bubble to rupture. The

mean velocity of the suspension travelling through the tube was approximately 0.0127 m/s, which

is obtained from flow rate measurements.

4.4 Future work

For the future work in the testing the capsules, there are a number of factors that can be tested. For

example one could test changing the transducer, either to a focused transducer, a transducer with

a different frequency or perhaps a clinical array transducer using multiple crystals. One could also

try using two transducers, one for firing at the suspension and another for visualization. Another

interesting change could be to try visualizing in real time. In this case, it would be required to have

a setup in which the microscope and transducer were aimed in the same direction and at the same

point.

14

5. Conclusion

The design setup aimed to investigate the effect of exposing a PFC5-suspension to ultrasound waves

with different acoustic properties. It also aimed to minimize the time between exposure and visu-

alising the suspension under a microscope. Volume distributions were generated by analyzing the

particle sizes in the microscopy images. In order to identify for which settings of ultrasound waves

ADV had occurred, the data was used to calculate mean and standard deviation values.

Evidence pointing towards ADV was found for two different combinations of ultrasound properties.

In both cases the ultrasound waves had a frequency of 3.5 MHz and a PRF of 500. The burst

width varied between the cases, being 12 cycles for one and 8 cycles for the other. For both cases

a significant increase in the standard deviation could be seen for PNP values ≥ 0.200 MPa. The

sudden increase of the standard deviation indicates that ADV has occurred, and that the pressure

threshold for ADV is located at 0.200 MPa for ultrasound waves with the mentioned properties.

The threshold was the same regardless if the burst width was set to 8 or 12. However the extent of

the ADV was larger when the number of cycles were higher.

The optimal PNP to promote ADV, for both aforementioned cases, seems to be for PNPs ranging

between 0.200 and 0.228 MPa. For higher PNP values the ultrasound has a reduced effect on the

distribution, possibly due the higher pressure leading to the destruction of capsules. The results from

other combinations of parameters tested in this study showed no clear evidence of the ultrasound

having any effect on the suspension.

Acoustic droplet vaporization has been shown to occur for different levels of acoustic power, indi-

cating the potential of the evaluated capsules as a PCCA as well as for therapeutic applications.

15

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18

A. State of the art

The aim of this project is to design a setup in which we can utilize a single crystal transducer to

attempt to induce Acoustic Droplet Vaporization (ADV) in a new type of Perfluoropentane (PFC5)

droplets and analyze their response. This state of the art report will act to convey the necessary

knowledge to perform and understand the project, including relevant research on the subject.

A.1 Ultrasound

There are several definitions which could accurately describe what ultrasound is, depending on what

field you study. For the purposes of this project however, the most fitting description would be that

ultrasound is mechanical waves with a frequency above 20 kHz.

A.1.1 Properties of the ultrasound wave

There is a great number of properties and characteristics associated with ultrasound and its uses,the

following are of extra importance within frame of the project.

A.1.1.1 Frequency

One very important parameter of ultrasound waves is the frequency, which in turns is directly

proportional to the wavelength. Even though the lower limit for ultrasound is 20 kHz, a much

higher frequency range of 2-15 MHz is commonly used clinically today [8]. The frequency is related

to wavelength according to the following formula:

f =c

λ(A.1)

Where λ is the wavelength and c is the speed of sound in the medium in which the wave is propogating

[8].

A.1.1.2 Acoustic Impedance

Acoustic Impedance is a material property which affects attenuation and reflection and is calculated

according to equation 2c below. The impedance of a material is related to the density and stiffness

1

of the material according to eq. 2a but is more commonly rewritten as eq. 2c with the help of

equation 2b [8].

z =√ρk (A.2a)

c =

√k

ρ(A.2b)

z = ρc (A.2c)

ρ is the density of the material and c is the speed of the sound in the material and k is the stiffness

of the material [8].

A.1.1.3 Attenuation

This brings up the concept of attenuation which is another very important phenomena within ul-

trasound physics. Attenuation is the loss of energy of the wave as it propagates through matter and

it depends on the impedance. The wave looses energy as it is converted into heat, this is known

as absorption, the material absorbs the energy [8].Reflection and scattering are other reasons for

attenuation in tissue, where parts of the amplitude is lost due to it changing direction [8].

A(z) = A0 · e−αz (A.3)

Equation 3 above is for calculating the attenuation of an ultrasound wave,where A0 is the initial

amplitude of the wave and α is what is known as the amplitude attenuation factor.

The attenuation is often measured in the amplitude decrease defined in decibels (dB) per centimeter,

dB cm−1, which is calculated as follows;

dBdecrease =1

z· 20 · log10(

A(z)

A0) (A.4)

A.1.1.4 Thermal and Mechanical index

The heat generated by attenuation could be potentially pose a problem in terms of safety, this and

the effects of the mechanical vibrations in tissue when the wave is propagating has given rise to

two properties that are also very important when using ultrasound, especially from a safety stand

point. These are Thermal index(TI) and Mechanical index (MI) which separately are measures for

the affect of the ultrasound on tissue.

The TI is a measure to quantify the risk of overheating a tissue, heating tissue can have serious

consequences if not kept to sufficiently low values.

TI =P

Pdeg(A.5)

Where P is the attenuated power at a specific location in the material and Pdeg is the power required

to raise the temperature of the material by 1 [8].

2

The MI is a similar measure to TI but instead of temperature it quantifies the effect of the movement

of tissue do the mechanical waves which are propagating through the tissue.

MI =PNP√Fc

(A.6)

Where PNP is the peak negative pressure of the ultrasound wave and is measured in MPa. Fc is

the center frequency of the wave which is measured in Mhz [8].

A.1.1.5 Burst width

The burst width in this case is defined as the number of cycles in each pulse, this is a dimensionless

property.

A.1.1.6 Repetition rate

The repetition rate affects at the rate of which pulses are fired from the transducer, measured in Hz

which is the same as pulses per second.

A.1.2 Transducer

In traditional ultrasound, a transducer is used to both send the ultrasound waves as well as listen to

the reflection of said wave. This works due to piezoelectric crystals that has the ability to convert

between electrical and mechanical energy by vibrating.

A.1.2.1 Single crystal transducer

For our purposes a single crystal transducer was used, this due to the increased flexibility in design-

ing the pulse sequence delivered and that a very precise calibration can be achieved with the help of

a hydrophone. The single crystal transducer offers a higher penetration and better uniformity then

the more clinically common array transducers which consists of an array of piezoelectric elements

instead of utilizing one element as for the single crystal transducer. There are two types of single

crystal transducers which are of interest; the flat transducer and the focused transducer.

For a flat transducer, the Near field is a parameter that needs to be taken consideration of. The

near field is the field after the transducer in which the size of the field is more or less unchanging in

size, the near field also has a complex pressure distribution. After the near field ends the far field

begins where the field will diverge, and the pressure distribution becomes more uniform the length

of the near field is obtained via:

N =d2

4λ=d2f

4c(A.7)

where d is the diameter of the beam, which is affected by transducer size [8].

3

As for the focused transducer, a single-crystal transducer achieves focus by creating a curved surface

of the transducer [8, 9] or by using an acoustic lens [8]. The focused transducer available for this

project has a curved surface.

A.2 Capsules

Central to this project are the capsules we are working with, the phase-changing contrast agents(PCCA).

The term ”capsule” is used here to incorporate both the droplet and bubble state of the PCCA, it

is sometimes useful to be able to refer to them as capsules when speaking of them in general and

not in which state they are in. PCCA are a certain type of contrast agent that is inactive when in

the ”droplet phase” and becomes an active contrast agent once the phase is changed to the ”bubble

phase”, hence the name. This is discussed more in section A.2.2.

A.2.1 Microbubbles

Microbubbles have been used as an ultrasound contrast agent(UCA) clinically for a number of years

now and has proven to be effective in certain applications, such as improving contrast and signal

strength from cerebral arteries [1]. Currently available MBs however have two major disadvantages

that limit their potential for use as contrast agents as well as their possibility for use in Ultrasound

Controlled Mediated Drug delivery. Firstly there is the relative short life time of the bubbles in

vivo, the bubbles will only remain for a matter of minutes before evaporating [3, 10, 11]. Secondly,

the size of the microbubbles confine them from entering extravascular space, limiting the UCA to

the vasculature and to use as a blood pool agent [1, 3, 12]. The size is also a limitation in the case

of drug delivery where the drugs will need to reach the intended targets of the drug.

When liquids are subjected to large variations in pressure a phenomena known as cavitation can

occur, where a sudden drop in pressure can cause cavities of vapor to form in the liquid. These

bubbles can collapse due to the pressure variations and the same goes for microbubbles in an acoustic

field. When the bubbles are in an acoustic field, the changes in pressure will cause the bubbles to

oscillate and, if the pressure is high enough, eventually burst creating a shockwave. This is called

inertial cavitation(IC), if the pressure is not high enough to cause the bubbles to collapse but to

oscillate the bubbles it is call non-inertial cavitation.

A.2.2 Acoustic droplets

These aforementioned limitations of currently available MB’s can be solved with the use of so called

Phase changing contrast agents which are contrast agents that are inserted into a patient as a liquid

filled bubble, an acoustic droplet, and subsequently forced to go through a phase change [3, 12]. This

is achieved via the use of ultrasound to increase temperature and pressure, reverting the bubble to a

gas-filled MB [3, 12]. The PCCA in droplet form has a smaller size than conventional MBs allowing

them to reach extra vascular spaces, and to ”extravasate through the leaky vasculature found in

malignant and inflamed tissues and can therefore enter diseased interstitial space” [3, 13, 2].They

also have a significantly longer lifetime in vivo, and the ability of reverting the droplets to a gaseous

MB allows them to then be used in both diagnostic and therapeutic capacities [3, 10, 12, 13].

4

The most common types of PCCAs are different types of perfluorocarbons (PFC), both with different

variations of the PFC and with different surfactants. Commonly the shell is made from phospho-

lipids or polymers [14, 15]. Experiments using bovine albumine as a shell has also been done [16].

As for the different types of PFCs used,in this application PFCs with low boiling points are con-

vention, many having boiling points below the temperature of the human body [14, 15, 16, 17].This

leads to the liquid within the capsules to be superheated which makes them ready to be instantly

evaporated when exposed to energy deposition from ultrasound, laser or any other source of energy.

The PFC used in this project was PFC5 which has a boiling point of 29 c◦ enveloped by a shell

made up of cationic cellulose nanofibers (CNF).

A.2.2.1 Manufacturing process of acoustic droplets

The acoustic droplets used in this study a new type of pickering stabilized perfluorodroplets. The

manufacturing of these droplets however complex from a chemical point of view, is quite straight

forward as to the lab work required. There are two main steps to the process, the first step is to

mix PFC5 with CNF. A scale is used to achieve correct weight ratio between the liquids, 1 g of

PFC5 is mixed with 36 g of 0.28 %wt CNF suspension.The mixture is then put on ice, to prevent

evaporation of PFC5, the mixture is subsequently sonicated for one minute at 80 % amplitude to

create the droplets incapsulated by CNF. The process has been described in more detail by other

sources [5]. The manufacturing and properties about these pickering emulsions and CNF-droplets

will not be discussed further in this paper, additional information can be found from the following

references [18, 19].

A.3 Acoustic Droplet Vaporization

The process of changing the phase of the capsules is commonly referred to as acoustic droplet vapor-

ization (ADV), it is also referred to as the activation of the PCCA. The result of ADV is that the

capsule goes from being an acoustic droplet to being a MB. This is achieved via ultrasound, which

alters pressure and temperature inside of the bubble to promote the vaporization of the PFC inside.

The exact mechanism behind it however is not fully understood. It was suspected that cavitation

was the underlying mechanism initiating the phase change, Kripfgans et al.(2000) however showed

that ”the acoustic pressure threshold for ADV decreases with increasing frequency, which is opposite

to what is known for the frequency-dependence of acoustic cavitation.” [16].Furthermore the MI of

the ultrasound wave decreases with increasing frequency [20]. This is important for clinical use of ul-

trasound and ADV, seeing as the MI of ultrasound applied to patients is restricted for safety reasons.

The threshold for acoustic activation of the PFC-droplets seem to be dependant on the the peak

of the negative half of the cycle of the acoustic wave or, as it commonly is referred to, the Peak

negative pressure (PNP) [21]. There is evidence that the nucleation of the droplets was induced at

the negative peak of the ultrasound wave [22].

When going through the phase change the droplets will not only change into bubbles, they will also

grow in size. Studies have shown that the capsules increase to about 4 to 6 times their original

size when ADV occurs [4, 21, 23, 24]. There is also evidence that the expansion can get as high as

10-fold in fluids which are not degassed, this due to gas diffusing from the surrounding fluid into

5

the capsule [4]. In 2014 it was observed that the size of the bubbles increased with longer pulse

duration, however an increase in acoustic power only had a small impact on the size distribution

[25]. The authors also observed that a suspension which had not been degassed resulted in larger

mean bubble size than when the suspension was in fact degassed [25].

A.3.1 Properties affecting ADV

Through the years of study on the vaporization of PFC-droplets some relationships that affect the

threshold of vaporization that have been found, namely studies have shown that an increase in ambi-

ent pressure will lead to an increase in the threshold, and inversely [17]. Increasing the temperature

will lower the threshold while decreasing the temperature will lead to a higher threshold [17, 26].

There is also evidence that an increase in the droplet diameter will lower the vaporization threshold

and vice-versa [17].

As mentioned above many kinds of PFCs have boiling points below the temperature of the body(PFC5

for example), and that it has been shown that the PFCs can exist ”in a superheated state without

spontaneous vaporization” when they are encapsulated into shell made of for example a polymer

[21]. This is due to the effect of Laplace pressure, which is given by the Young-Laplace equation

[21].

∆P = γ · ( 1

R1+

1

R2) (A.8)

where γ is the surface tension and R is the radius of the droplet [27]. For droplets or bubbles

R1 = R2 = R and the equation simplifies to ∆P = γ · 2R

The boiling point of the PFC will also affect the threshold for vaporization [3].

A.3.2 Effects of constrained geometry

In this project a tube with a diameter of 500 µm is used to contain and transport the capsules, the

capsules are therefore in this confined space when the ultrasound is applied and ADV is initiated.

There have been studies on the effects of constraining geometry on ADV as well as microbubble

oscillations.Experiments on bubble growth in tubes with sizes of 12 µm, 25 µm and 195 µm have

been performed, and showed that microbubbles confined in a tube with a diameter in the order of

the microbubbles themselves expanded less than microbubbles which were confined in tubes with a

greater diameter [28].

Additional research on the effects of constraining gemoetry has been done, for example research to

compare contrast enhancement of PCCAs in an open environment compared to when confined by

a 200 µm tube. The conclusion reached was that the contrast enhancement was reduced 19.5-fold

for Pulse inversion signals and with a factor of 24.7 for B-mode signals for the confined PCCAs,

indicating the constrained geometry having a significant impact on the ADV of the PCCAs [12].

Another paper published in 2013 compared oscillations for MBs in a 160 µm tube with those in a 25

µm tube. The result showed that the there was of up to 50 % more oscillations in the bigger tube

than in the smaller one [29].

6

A.4 Potential areas of use for ADV

The potential of use for ADV is large, the process of ADV can improve most of the application of

microbubbles with respect to ultrasound. This section will go through some of the more promising

areas of use.

A.4.1 Contrast agents

A commonly occurring clinical use for MBs is as a contrast agent for ultrasound imaging, therefore

the use of ADV within contrast agent is an important aspect [30]. The foremost advantage of em-

ploying acoustic droplets and ADV for contrast purposes is the increased lifetime of the droplets,

which can be used to provide contrast for a longer of period of time at the areas of interest by not

activating the capsules until they are ready to image.

It has been that ADV-generated microbubbles increased the SNR when imaging with B-mode ul-

trasound, this indicates that ADV can be a useful tool as an improved contrast agent [31]. More

specifically ADV is especially useful when correcting ”Transcranial ultrasound phase aberrations”

as is explained in the following papers [32, 33].

A.4.2 Drug delivery

The combination of US and MB have fairly recently emerged as an interesting alternative for drug

delivery, which is an upcoming field within ultrasound technology. The use of these techniques for

drug delivery show a lot of promise and could have a big impact on the outcome of for example

chemotherapy [2].

The advantage being that the droplets can present a way of transporting drugs across biological

barriers to location where drugs otherwise cannot enter, such as the Blood Brain Barrier [34].

These applications within drug delivery is very prevalent in cancer treatment, ADV could be useful

in several ways for treating malignant tumours. Firstly the growing capsules could be used to block

the blood supply of tumours by obstructing the vessels with bubbles, this is called vascular occlu-

sion. Furthermore ADV could be used to deliver e.g chemotherapy drugs directly into the tumours,

these two techniques can also probably be used in tandem to help increase the effectiveness of killing

tumours [35].

Another way in which ADV can help in affecting tumour treatment is via what is known as ablation,

which is damages caused by cavitation of microbubbles, ADV can help in increasing the damage

caused by increasing the number of cavitation nuclei [36].

The biophysical mechanism that enables enhancement of drug delivery by utilizing ultrasound to

mediate the delivery are not fully understood or agreed upon in the field [37]. Sonoporation is the

most common theory for the main reason behind the phenomena [37].

A number of studies have been made recently to increase the knowledge of these microbubbles as

well as on the prospect of using them in drug delivery. One recent study evaluated MBs from a

theranostic perspective in which the used high power ultrasound to achieve an US triggered release

7

of nitric oxide in vitro, with MBs loaded with nitric oxide [38].

I has been showed that when utilizing ADV for drug delivery there was two main factors that

increased the efficacy of the tumour therapy, the effect of the locally delivered drugs as well as the

tumour tissue destruction caused by the inertial cavitation of the capsules [39].In this paper they

used HIFU to initiate ADV, HIFU is another field in which ADV can be of use, this is explained

further in the next section. Further studies also observed the effects of ADV on the efficacy of drug

delivery and showed that ADV improved extravasation due to vessels being disrupted [40].

Capsules containing PFC5 which where vaporized could merge into larger bubbles after activation.

With sufficient pressures it was observed that IC could be induced. Above it was mentioned that

cavitation can increase the effect of drug delivery due to the disruption of vessels, as mentioned

above. This together with the fact that the merging of bubbles can aid the blockage of blood flow

into tumours makes ADV-based drug delivery a promising field [31].In this study droplet-loaded

macrophages was used to deliver the drugs.

A.4.3 HIFU treatment

ADV has shown promise in the field of High Intensity Focused Ultrasound (HIFU) treatment to help

enhance the effect. When comparing HIFU combined with ADV to HIFU alone for in-vivo tumours

there was a 2.9-fold increase in necrosis of tissue and the volume of malignant tissue was decreased

with a factor of 30 when ADV was introduced [23].

It has also been show that HIFU can be used to enable drug delivery into solid tumours, as mentioned

earlier this is due to the disruption of vessels and the increased cellular permeability [40, 41].

A.4.4 Safety

As has been mentioned above, there are a lot of uses for ADV in combination with ultrasound.

However there has been suspicion that the process of ADV could potentially be harmful, especially

when it occurs in very small capillaries.

There has been experiments conducted with ADV on ”vessel-mimicking phantoms” and found that

ADV lead to the creation of lesions on the phantom walls [21]. Further research shows that ADV

”near the endothelium may impact the vessel wall”, this effect can be both negative and positive

[24]. The effect is positive if the aim is to e.g treat malignant tumours, then the damage done can

be beneficial, however if the aim is to use ADV in contrast purposes, the effect is a negative one.

The change of volume of the capsules could also lead to injuries on cells due to the sudden increase

of pressure and stress caused by the capsule expanding inside of the blood vessel [24]

The possible safety issues involved with ADV mentioned above is something that needs to be taken

into consideration when ADV-related techniques start to be performed in-vivo.

8

Appendix B: Volume distributions

This appendix presents all the Volume distribution plots that were gathered from the experiments

but not included in the thesis.

9

((a)) Reference with no ultra-sound activated(before)

((b)) Reference with no ultra-sound activated(after)

((c)) 0.144 MPa ((d)) 0.169 MPa

((e)) 0.200 MPa ((f)) 0.228 MPa

((g)) 0.260 MPa ((h)) 0.291 MPa

Figure B.1: Volume distribution for PRF=500 and Cycles=8 for 0.244 to 0.291 MPa PNP.

10

((a)) Reference with no ultrasoundactivated(before)

((b)) Reference with no ultrasoundactivated(after)

((c)) 0.144 MPa ((d)) 0.260 MPa

((e)) 0.228 MPa ((f)) 0.200 MPa

((g)) 0.260 MPa ((h)) 0.291 MPa

Figure B.2: Volume distribution for PRF=500 and Cycles=4 for 0.144 to 0.291 MPa PNP

11

((a)) Reference with no ultrasoundactivated(before)

((b)) Reference with no ultrasoundactivated(after)

((c)) 0.144 MPa ((d)) 0.169 MPa

((e)) 0.200 MPa ((f)) 0.228 MPa

((g)) 0.260 MPa ((h)) 0.260 MPa

Figure B.3: Volume distribution for PRF=100 and Cycles=12 for 0.144 to 0.291 MPa PNP

12

((a)) Reference with no ultrasoundactivated(before)

((b)) Reference with no ultrasoundactivated(after)

((c)) Volume distribution with 0.144MPa PNP

((d)) Volume distribution with 0.169MPa PNP

((e)) Volume distribution with 0.200MPa PNP

((f)) Volume distribution with 0.228MPa PNP

((g)) Volume distribution with 0.260MPa PNP

((h)) Volume distribution with 0.291MPa PNP

Figure B.4: Volume distribution for PRF=500 and Burst width= 12 for PNP ranging from 0.144to 0.291 MPa.

13

Appendix C: Mean value plots

This appendix presents all the mean value plots that were gathered from the experiments but not

included in the thesis.

Figure C.1: Plot of the mean diameterfor each level of acoustic power for burst

width=12and PRF=500

Figure C.2: Plot of the mean volumefor each level of acoustic power for burst

width=12 and PRF=500

Figure C.3: Plot of the mean diameterfor each level of acoustic power for burst

width=8 and PRF=500

Figure C.4: Plot of the mean volumefor each level of acoustic power for burst

width=8 and PRF=500

Figure C.5: Plot of the mean diameterfor each level of acoustic power for burst

width=4 and PRF=500

Figure C.6: Plot of the mean diameterfor each level of acoustic power for burst

width=4 and PRF=500

1

Figure C.7: Plot of the mean diameterfor each level of acoustic power for burst

width=12 and PRF=100

Figure C.8: Plot of the mean volumefor each level of acoustic power for burst

width=12 and PRF=100

2

3

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