Microbubble Symposium: Fabrication, Characterisation and ...

Microbubble Symposium: 

Fabrication, Characterisation and Translational 

Applications 

16 th & 17 th July 2012 

Weetwood Hall, Otley Road, Headingley, Leeds LS16 5PS

2 Microbubble Symposium: Fabrication, Characterisation and Translational Applications 

16 th & 17 th July 2012

Microbubble Symposium: Fabrication, Characterisation and Translational Applications 3 

16 th & 17 th July 2012 

Contents 

Program 4 

Abstracts: 

Page 

Oral presentation – Session 1 

Microbubble Fabrication and Characterisation 6 


Microbubble Ultrasound Characterisation 12 


Microbubble Translational Applications 16 

Poster presentations 22 

Delegate list 49


16 th & 17 th July 2012 

Monday 16 th July 2012 

Program 

13:00 – 14.00 Registration 

14:00 – 14:10 Stephen Evans Welcome 

Session 1: Microbubble Fabrication and Characterisation – Chair: Stephen Evans 

14:10 – 14:50 Nico de Jong Microbubbles for Medical Use 

14:50 – 15:30 Mohan Edirisinghe Preparation of Bubbles and Capsules: 

Electrohydrodynamics vs Microfluidics 

15:30 – 15:55 Radwa Abou-Saleh & 

Sally Peyman 

15:55 – 16:30 Tea / Coffee 

High On-Chip Production Rates and Mechanical 

Characterisation of Therapeutic Microbubbles 

for Targeted Drug Delivery. 

16:30 – 17:10 Vasileios Koutsos Nanomechanical Properties of Microbubbles 

Studied by Atomic Force Microscopy 

17:10 – 17:35 Jonathan Loughran Investigation into the Effects of Ultrasound on 

Attached Microbubbles 

18:00 – 19:15 Drinks and Posters 

19:15 – 21:15 Symposium Dinner 

21:15 - onwards Informal discussions in the Stables pub


16 th & 17 th July 2012 

Tuesday 17 th July 2012 

Session 2: Microbubble Ultrasound Characterisation – Chair: Steven Freear 

09:00 – 09:40 Paul Dayton Microbubble Imaging in Cancer Theranostics 

09:40 – 10:20 John Hossack Catheter-Based Monodisperse Microbubble 

Production for Image-guided Vascular Therapy 

10:20 – 10:50 Tea / Coffee 

10:50 – 11:15 James McLaughlan The Acoustic Evaluation of Liposome-Loaded 

Microbubbles for Targeted Cancer Therapy 

11:15 – 11:55 Ayache Bouakaz Ultrasound Contrast Agents: from Imaging to 

Therapy 

11:55 – 13:30 Lunch 

Session 3: Microbubble Translational Applications – Chair: Louise Coletta 

13:30 – 14:10 Carmel Moran Characterisation of Ultrasonic Contrast 

Microbubbles for Preclinical Applications 

14:10 – 14:50 Jithin Jose Multi-modal Contrast Agents: Photoacoustic 

Imaging and Therapy 

14:50 – 15:15 Nicola Ingram Microfluidically-Generated Microbubbles for 

Molecular Imaging and Drug Delivery in 

Colorectal Cancer – Pre-Clinical Evaluation 

15:15 – 15:45 Tea / Coffee 

15:45 – 16:25 Robin Cleveland Therapeutic Uses of Microbubbles: Heating, 

Erosion and Delivery 

16:25 – 17:00 Jane Smith Contrast Enhanced Ultrasound in Clinical 

Practice 

17:00 – 17:10 Sir Alex Markham Closing remarks


16 th & 17 th July 2012 

Session 1: Microbubble Fabrication and Characterisation 

Nico de Jong 

ErasmusMC, Rotterdam and University Twente 

Oral Presentation 

Title: Microbubbles for Medical Use 

Abstract: Ultrasound is the most widely used medical imaging modality. It is capable of providing real-time 

information about tissue structures and blood flow in the heart and larger vessels. For determining perfusion 

Ultrasound Contrast Agents (UCA) are mandatory. Besides using UCA for measuring perfusion there is a 

growing interest in the use of coated microbubbles for therapeutic applications. In this case the microbubbles 

can act as transport carriers for drug delivery or when targeted, to image the targeted site, referred as 

molecular imaging. A third application for therapy is known as sonoporation. Here, the oscillation of 

ultrasound-driven microbubbles, in close contact with a cell, lead to an increased permeability of the cell to 

macromolecules, hence to an increased uptake of drugs or genes. 

In ultrasound contrast imaging one utilizes the non linear vibrations of the bubbles in the ultrasound field. 

When the generated ultrasound wave hits a free or encapsulated microbubble, a volume pulsation is initiated. 

Depending on the magnitude of the ultrasound wave the pulsation will be: linear to the applied pressure or 

non-linear to the applied pressure. Non linear vibration can be split into: stationary (harmonic) and transient 

(power) scattering. Depending on the agent three acoustic pressure regions can be distinguished: linear 

scattering: 0-50, harmonic scattering: 50-200 and transient power scattering: 200-2000 KPascal. 

Bubble vibration can be seen under the microscope and a ultrafast framing camera. A high frame rate is 

mandatory to record the vibration of a bubble when it is insonified with 2-3 MHz ultrasound, which is the 

normal diagnostic frequency. The figure below presents an example of a recording with such a camera 

(Brandaris 128) showing an isolated bubble with a size of 4.2 µm in diameter that is insonified with an acoustic 

pressure of 250 kPa at a frequency of 1.7 MHz. In 64 frames we observe the bubble before, during and after 

the ultrasound pulse. In the first 13 frames, the microbubble is at rest. Starting at frame 14, the microbubble is 

first compressed, and then reaches within six cycles a maximum diameter of 5.4 μm and a minimum diameter 

of 2.6 µm. We can establish in each image frame the bubble diameter. The resulting diameter-time (D-T) curve 

is shown in the panel in the middle and the corresponding power spectrum in the right panel. 

With the camera we have observed numerous phenomenon, 

like subharmonics, ultraharmonics, thresholding, 

compression only, expansion only, thresholding and mode 

vibration as shown in the figure. Moreover, the behaviour of 

an individual bubble nearby a wall or cell has been 

extensively examined to resolve the mechanism behind 

sonoporation and binding. 

The Frequency power 

spectrum shows directly that 

the bubble not only scatter 

the fundamental frequency at 

1.7 MHz, but also a second 

harmonic component at 3.4 

MHz. The level of this second 

harmonic is about -17 dB 

below the fundamental. This 

harmonic vibration amplitude 

generates a pressure wave 

which is about 50 % of the 

fundamental pressure wave.


16 th & 17 th July 2012 



Mohan Edirisinghe 

University College London 

Title: Preparation of Bubbles and Capsules: Electrohydrodynamics vs Microfluidics 

Abstract: Two major methods gathering tremendous interest for the mass production of 

microbubbles and micro/nano capsules are co-axial electrohydrodynamic microbubbling 

(CEHD-M) and T-junction microfluidics (TJM). This lecture will summarise the key recent 

developments in these techniques, mainly focussing on efforts to control size and size 

distribution of the products. In the case of CEHD-M the use of >2 needles has been a major 

feature 1 . Regarding TJM, the junction geometry, its dimensions and the central zone where 

fluids meet and interact has been fundamentally changed. These changes have resulted in 

several new products 2 and, also led to the controlled preparation of nanoparticles from 

bubbles directly at the microfluidic junction site, a very useful feature for drug delivery 3 . 

References: 

1) M-w.Chang, E.Stride and M.Edirisinghe, Layered Bodies, Compositions Containing Them and Processes 

for Producing Them, PCT/GB2012/050276, 8 th Feb. 2012. 

2) O.Gunduz, Z.Ahmad, E.Stride and M.Edirisinghe, Mater. Sci. Eng. C, 32(2012)1005-1010. 

3) O.Gunduz, Z.Ahmad, E.Stride, C.Tamerler and M.Edirisinghe, J. Royal Soc. Interface, 9(2012)389-395.


16 th & 17 th July 2012 



Radwa Abou-Saleh & Sally Peyman 

University of Leeds 

Title: High On-Chip Production Rates and Mechanical Characterisation of Therapeutic 

Microbubbles for Targeted Drug Delivery. 

Abstract: Micron sized, lipid stabilized bubbles of gas are of interest as contrast agents for 

ultra-sound (US) imaging and increasingly as vehicles for targeted drug delivery. 

Microfluidics provides a reproducible means for microbubble production and surface 

functionalisation using flow-focussing microfluidic devices that combine streams of gas and 

liquid through a nozzle a few microns wide and then subjecting the two phases to a 

downstream pressure drop. While microfluidics has successfully demonstrated the 

generation of monodisperse bubble populations, these approaches inherently produce low 

bubble counts. We introduce a new micro-spray flow regime that generates consistently 

high bubble concentrations, in the order of 1010 bubbles mL-1. The technique is shown to 

be easy to implement and highly reproducible. We also demonstrate that it is possible to 

attach particles such as liposomes, loaded with quantum dots or fluorescein in a single step 

during the MB formation procedure. 

The frequency response of these microbubbles to ultrasound excitation mainly depends on 

the mechanical properties of the shell which can be determined using atomic force 

microscopy (AFM). The addition of different components to the shell architecture can 

change the bubbles mechanical properties and therefore its ultrasound response. We 

present force spectroscopy measurements with tip-less cantilevers, on microbubbles, to 

characterize the shell stiffness of a range of different shell coatings, specifically, Streptavidin, 

Q-dots, and liposomes. Forces ranging from 20-100 nN were applied to single bubbles to 

determine the change in stiffness with applied force. It was observed that the shell stiffness 

of phospholipid microbubbles increases linearly with increasing applied force. Adding a 

Streptavidin coat doubles the stiffness values of the microbubble, with a dramatic three-fold 

increase in shell stiffness observed with addition of Q-dots.


16 th & 17 th July 2012 



Vasileios Koutsos 

University of Edinburgh 

Title: Nanomechanical Properties of Microbubbles Studied by Atomic Force Microscopy 

Abstract: Stable, haemodynamically inert, hollow, micrometer-size spheres composed of an 

ultrathin nanoscale biocompatible shell which encapsulates an inert gas are used as ultrasound 

contrast agents and are normally referred to as microbubbles. They are smaller than the smallest 

blood vessel of a human body to allow improved visualization of the vascular bed and differentiate 

vascular patterns of tumours non-invasively. Such thin-shelled microstructures can be used as 

carriers for drug/gene delivery, which is a topic of much current interest in biomedical research. 

Furthermore, with appropriate surface modification such microbubbles can acquire targeting 

capability for certain cell types (e.g. cancerous cells). Their nano/micromechanical properties are 

extremely important since they have to be stable for considerable time until they rupture/degrade 

under specific conditions in order to release their load in the right place and at the right time. 

Moreover, materials at the nanoscale (such as thin-shell structures) may behave differently to those 

on the macroscale, so predicting their mechanical properties presents a challenge. We have 

conducted a systematic study of both hard-shelled, polymeric-based 1,2 and soft-shelled, 

phospholipid-based 3 microbubbles employing microcompression by performing atomic force 

microscopy force-distance curves. Individual microbubbles of different diameter were compressed 

from few nanometers up to approx. 50% of their initial diameter. We have performed multiple 

compressions on individual microbbubles to assess their mechanical robustness and measured their 

stiffness. Furthermore, we used different mechanical theories to estimate the elastic modulus of the 

microbubble shell. In the case of phospholipid-based shells, we have also estimated the effective 

elastic modulus of the whole microbubble as (if it was) a homogenous object. We compare and 

discuss our results with other phospholipid systems, namely supported lipid membranes, vesicles and 

cells and other recent AFM studies of lipid-coated microbubbles. 4,5 Furthermore, as microbubble 

science is expanding to biological targeting and drug/gene delivery, we investigated the 

characteristics of molecular targeting using modified lipid-shelled microbubbles (biotin–avidin 

chemistry and the CD31 antibody) to probe individual Sk-Hep1 hepatic endothelial cells. The 

modified (targeted) microbubbles provide a single distribution of adhesion forces with a median of 

93 pN. This interaction was assigned to the CD31 antibody–antigen unbinding event and proves the 

capability of single microbubbles to target cell lines. 6 

References: (1) E. Glynos, V. Koutsos, W. N. McDicken, C. M. Moran, S. D. Pye, J. A. Ross, V. Sboros. 

Nanomechanics of Biocompatible Hollow Thin-Shell Polymer Microspheres. Langmuir 2009, 25 (13), 7514−752; 

(2) E. Glynos, V. Sboros, V. Koutsos. Polymeric thin shells: Measurement of elastic properties at the nanometre 

scale using atomic force microscopy. Materials Science and Engineering: B 2009, 165 (3), 231−234; (3) E. 

Buchner Santos, J. K. Morris, E. Glynos, V. Sboros, V. Koutsos. Nanomechanical Properties of Phospholipid 

Microbubbles. Langmuir 2012, 28 (13), 5753−5760; (4) J. E. McKendry, C. A. Grant, B. R. G. Johnson, P. L. 

Coletta, J. A. Evans, S. D. Evans. Force spectroscopy of streptavidin conjugated lipid coated microbubbles. 

Bubble Sci., Eng., Technol. 2010, 2 (2), 48−54; (5) C. A. Grant, J. E. McKendry, S. D. Evans. Temperature 

dependent stiffness and visco-elastic behaviour of lipid coated microbubbles using atomic force microscopy. Soft 

Matter 2011, 8, 1321−1326; (6) V. Sboros, E. Glynos, J. A. Ross, C. M. Moran, S. D. Pye, M. Butler, W. N. 

McDicken, S. B. Brown, V. Koutsos. Probing microbubble targeting with atomic force microscopy. Colloids and 

Surfaces B: Biointerfaces 2010, 80 (1), 12−17


16 th & 17 th July 2012 



Title: Investigation into the Effects of Ultrasound on Attached Microbubbles 

Jonathan Loughran 

Imperial College London 

Abstract: Microbubble contrast agents have shown great potential in imaging and 

quantifying blood flow and tissue perfusion. Recent advance of targeted microbubbles have 

made it possible to detect and imaging the distribution of specific molecules of interest 

expressed on endothelial walls in vivo using ultrasound. Previous studies have been focused 

on developing microbubbles with improved targeting efficiency, acoustic techniques to 

facilitate the attachment of such bubbles and better detection method, little attention has 

been paid to study what happens to the microbubbles once they are attached. In this study 

the aim is to study the effects of ultrasound exposure to attached microbubbles. Biotin 

coated microbubbles were flown through a phantom coated with streptavidin. Optical 

microscopy was used to observe the attached microbubbles in the flow phantom under 

ultrasound exposure. The bubble size and position were recorded during the exposure of 

ultrasound. It was found that even at relatively low acoustic pressure a significant amount of 

bubbles detach from the wall and flow away, some of them shrink at the same time. The 

number of bubble detachment increases with acoustic pressure. The different forces acting 

on the bubbles during the exposure was analysed to offer some insights into the detachment 

mechanisms. 

Additional authors: 

Jonathan Loughran1, Charles Sennoga1, Robert Eckersley2, Meng-Xing Tang1* 

1 Department of Bioengineering, Imperial College London, London SW7 2AZ, UK 

2 Biomedical Engineering Department, King's College London, London. SE1 7EH, UK


16 th & 17 th July 2012


16 th & 17 th July 2012 

Session 2: Microbubble Ultrasound Characterisation 


Title: Microbubble Imaging in Cancer Theranostics 

Paul A Dayton 

University of North Carolina 

Abstract: Theranostics refers to the combination of diagnostic and therapeutic approaches, 

typically for assessing response to therapy. Although it is possible for an agent to be used 

simultaneously for imaging and therapy, by definition, theranostics is fundamentally the 

combination of a diagnostic test with a therapeutic approach, and thus can also involve 

either application of a diagnostic technique followed by a therapeutic, or vice versa. 

Theranostic approaches are a key aspect in the drive toward personalized medicine, where 

the tailoring of decisions and treatment practices for individual patients are based on 

diagnostic information specifically from that patient. Microbbubles can be used for imaging, 

for therapy, or both simultaneously. In this sense, microbubbles combined with acoustics 

may be one of the most universal theranostic tools. In this seminar, we will specifically focus 

on microbubble contrast agents and contrast imaging techniques for assessment of tumor 

growth and response to therapy. Both perfusion imaging and molecular imaging are shown 

to differentiate differences between tumors which respond to therapy and those which do 

not, at time points earlier than determined by traditional tumor size measurements. 

Contrast enhanced microvascular mapping enables the delineation of tortuous tumor 

microvasculature associated with angiogenesis. When analyzed with segmentation 

techniques, this method can also indicate malignancy based on microvascular structure 

alone. These results suggest that contrast enhanced ultrasound may play a substantial role 

in noninvasive assessment of tumor response to therapies and personalized medicine.


16 th & 17 th July 2012 



John A. Hossack 

University of Virginia 

Title: Catheter-Based Monodisperse Microbubble Production for Image-guided Vascular 

Therapy 

Abstract: Current methods for delivering smooth muscle cell antiproliferative drugs to the vessel 

wall following percutaneous-transluminal angioplasty and stent placement are limited in terms of 

ability to tailor drug choice and delivery profile to a particular patient’s lesion. We have been 

investigating intravascular ultrasound (IVUS)-based methods for imaging and drug delivery using 

ultrasound in combination with drug associated microbubbles. Following recognition in the field that 

precise control of both ultrasound parameters and microbubble properties are preferable to achieve 

efficacy and safety simultaneously, we have been investigating microfluidics-based approaches to 

microbubble production that yield mono-disperse microbubbles. In this paper, we present a new 

method for supplying the liquid (shell) phase to a flow focusing microfluidic device (FFMD). In place 

of individual tubes supplying each inlet, the FFMD is coupled to a pressurized liquid-filled chamber 

that drives the fluid from inside the catheter directly into the channels of the microfluidics device. 

This method eliminates bulky interconnects, significantly reducing the complexity of the device and 

simplifies device parallelization enabling increased microbubble production rate and distributed 

production around the circumference of the catheter. Flooded FFMDs were fabricated and 

production properties quantified. The minimum microbubble size produced was 8.1±0.3 µm. The 

maximum production rate from a single nozzle FFMD was 660,000 microbubbles/s. Production was 

1.5-fold higher when using a parallel device. Microbubbles produced in situ in a gelatin flow phantom 

were imaged in real time using a co-aligned IVUS and an external linear array clinical scanner 

demonstrating contrast enhanced imaging of the vessel. In addition, delivery of a co-injected dye was 

enhanced in areas subject to both microbubbles and ultrasound. Therefore, in situ produced 

microbubbles, coupled to an IVUS catheter, have the potential to contribute to the long-term goal of 

providing real time image guided diagnosis and treatment of vascular disease. 

(a) 

(b) 

(c) 

(a) Schematic of an IVUS catheter 

functionalized with a flooded FFMD 

for producing mono-disperse 

microbubbles in situ. (b) IVUS image 

of vessel lumen enhanced with realtime 

production of microbubbles. (c) 

Enhanced delivery of a fluorophore to 

the vessel wall subject to both 

ultrasound and microbubbles


16 th & 17 th July 2012 



James McLaughlan 


Title: The Acoustic Evaluation of Liposome-loaded Microbubbles for Targeted Cancer 

Therapy 

Abstract: The use of microbubbles as drug or gene delivery vehicles is a highly active area of research, as they 

can be targeted to specific cell populations delivering therapeutic agents to a localised area. Attachment of 

drug filled liposomes to the outside of microbubbles is one mechanism. The attachment of liposomes onto the 

shell can affect their ultrasound response, which is important for both imaging and therapeutic applications as 

it could result in expansion only oscillations and/or increasing the destruction threshold. 

Phospholipid microbubbles (1-10 µm), manufactured in a microfluidic flow cell, were conjugated with 

fluorescein filled liposomes (200 nm). A 4 MHz windowed tone burst was used to excite a microbubble 

population (0.5x10 6 MB/ml) and the 90° scatter was detected using a 1 mm needle hydrophone. The 

destruction threshold was determined in a flow cell mounted on top of an inverted microscope. Bright field 

images taken before and immediately after the ultrasound exposure were used to estimate change in 

population for a given mechanical index (MI). The acoustic response and destruction threshold was measured 

with and without attached liposomes. 

The figure below, (a) shows bright field and fluorescent images of loaded and unloaded microbubbles. Only the 

loaded microbubbles are visible under fluorescence due to the green fluorophore encapsulated within the 

liposomes. Fig. (b) and (c) show the scattered frequency spectrum from a population of microbubbles at 50 and 

300 kPa (MI 0.025 and 0.15). The subharmonic ratio shown in (d) demonstrates that for low MI the loaded 

microbubbles have a significantly larger subharmonic emission than the unloaded microbubbles. Once an MI of 

0.1 is exceeded the unloaded microbubbles increase in subharmonic emission, however as (e) and (f) show, this 

could be due to microbubble destruction and release of free gas bubbles. The liposome loading has a negligible 

effect on the destruction of microbubbles since (e) and (f) show that for both types 90% of the population is 

destroyed for a MI of 0.4. 

Liposome loading of microbubbles causes an increase in their harmonic response, specifically the subharmonic, 

for low ultrasound pressures (MI). Once microbubble destruction is present the acoustic response can no 

longer be attributed to the shell of the microbubble, since free gas bubbles could also be present.


16 th & 17 th July 2012 



Title: Ultrasound Contrast Agents: from Imaging to Therapy 

Ayache Bouakaz 

Université François Rabelais, Tours 

Abstract: Contrast agents, consisting of tiny gas microbubbles are currently approved for 

ultrasound imaging in cardiology and in radiology. The microbubbles have a mean size of 

about 3 microns and are encapsulated by a thin biocompatible layer. Multiple clinical studies 

have established the utility of ultrasound contrast agents (UCA) in improving accuracy of 

echography for the diagnosis of many diseases and in reducing health care costs by 

eliminating the need for additional testing. 

Future clinical applications of UCA extend beyond imaging and diagnostic, offering to 

ultrasound technology a new therapeutic dimension. Since a few years, novel therapeutic 

strategies are explored using microbubbles and ultrasound. Current data demonstrate that 

in the presence of microbubbles, ultrasound waves destabilize transiently the cell membrane 

allowing the incorporation of drugs, including genes into the cells. Moreover, the 

microbubbles might be used as a drug vehicle to achieve a spatially and temporally 

controlled local release. Besides, microbubbles are able to identify diseased targets through 

specific targeting. The talk will focus on the current knowledge of microbubble targeted 

therapy fundamentals and basic mechanisms. New challenges will be discussed.


16 th & 17 th July 2012 

Session 3: Microbubble Translational Applications 


Carmel M Moran 

University of Edinburgh 

Title: Characterisation of Ultrasonic Contrast Microbubbles for Preclinical Applications 

Abstract: Research using small animals, continues to play a key role in the advancement of 

medical, biological and veterinary sciences. In particular the mouse model is popular as a 

research model as over 85% of the genomic sequences in mice are identical to those found 

in humans. In addition, the use of zebra-fish (Danio rerio) has increasingly become an 

important tool in medical research with application to the investigation of an extensive 

range of human diseases. Its small size and corresponding small space requirements, 

relatively rapid sexual maturity (approximately 3 months), generation of hundreds of 

embryos every week and external fertilisation makes it increasingly one of the most 

accessible animals for developmental studies. The use of high resolution ultrasound coupled 

with light anaesthesia ensures that preclinical ultrasound is a relatively inexpensive , high 

throughput technique useful for determining the phenotype of these animals. Ultrasonic 

contrast microbubbles, in particular targeted agents are increasingly used to deliver either 

genes or drug payload to specific sites within these animals. 

In this presentation I will focus on our work on measuring the acoustic properties of 

contrast agents at frequencies used for preclinical imaging and the implications of our 

results on in-vivo animal imaging. We use a Vevo 770 (Visualsonics Inc, Toronto) and four 

transducers (RMV 710, RMV 707, RMV 704 and RMV 711) and employ a broadband 

substation technique to measure the acoustic properties of tissue-mimicking-material 

(TMM) and three commercially available contrast agents (SonoVue, Definity and 

Micromarker) over the frequency range from 10-47MHz. Specficially, we have measured 

the contrast-to-TMM ratio and the attenuation of the contrast agents at non-destructive 

pressures. Our preclinical studies have focussed on the use of commercially available 

clinical contrast agents and the settings required to optimise these agents for visualisation 

within our preclinical models.


16 th & 17 th July 2012 



Title: Multi-modal Contrast Agents: Photoacoustic Imaging and Therapy 

Jithin Jose 

Visualsonics 

Abstract: Photoacoustic (PA) imaging is a hybrid imaging modality, for non-invasive 

detection of tissue structural and functional anomalies. The technique combines the 

advantageous properties of optical and ultrasound imaging. It provides the excellent 

contrast achieved in optical techniques with the high spatial resolution of ultrasound 

imaging. Some important applications of this technique include breast cancer detection, skin 

cancer visualization and small animal imaging. Multifunctional microbubbles (MBs) are 

contrast agents integrating multiple disease‐targeting, imaging and therapeutic functions. 

Multifunctional MBs represent an enabling technology for many potential applications in the 

field of photoacoustic imaging. Highly absorbing optical contrast agents are encapsulated in 

the bubbles and it can be used as multi-modal contrast agents for high resolution ultrasound 

and photoacoustic imaging. Upon activation the bubbles may introduce significant contrast 

enhancement and facilitate a variety of potential clinical and preclinical applications 

including photo thermal therapy.


16 th & 17 th July 2012 



Nicola Ingram 


Title: Microfluidically-Generated Microbubbles for Molecular Imaging and Drug Delivery in 

Colorectal Cancer – Pre-Clinical Evaluation 

Abstract: Drug delivery to solid tumours and metastatic lesions remains a major problem in 

the treatment of cancer. Ultrasound (US) contrast agents or microbubbles (MBs) are 

currently being developed for their theragnostic capabilities, potentially linking US diagnosis 

with drug delivery and monitoring of therapeutic response. To this end, the Leeds 

Microbubble Consortium has developed a novel on-chip flow focusing microfluidic (µF) 

device capable of reproducibly generating MBs in sufficient quantity and quality for use in 

vivo. 

We have performed extensive in vitro and in vivo evaluation of our µF MBs to analyse their 

performance in US imaging and targeted drug delivery. Increased µF MB lifetime in vitro 

was shown to be dependent on shell lipid concentration. Toxicity was assessed in vitro and 

found to be comparable to Micromarker (Bracco). In vitro binding to endothelial and 

colorectal cancer (CRC) cells using µF MBs conjugated with anti-VEGFR2 antibodies was 

examined by cell flow assays. Significantly higher binding was found with VEGFR2 positive 

cells compared with negative cells or with µF MBs conjugated to isotype control antibodies. 

In vivo imaging in mouse colorectal cancer models with µF MBs showed clear contrast of the 

vascular space and generated wash-in curves for quantitative evaluation. 

We have also studied the spatial and temporal expression of VEGFR2 in a CRC model to 

determine its suitability for molecular imaging and targeted delivery. Using histological 

methods, we have shown a peak in CD31+ blood vessels that also expressed VEGFR2 at 

approximately 50mm 3 in size. Expression then decreased as the tumours increased in 

volume. This was comparable with the signal obtained from VEGFR2-targeted MBs in vivo 

where smaller tumours demonstrated a greater subtracted molecular signal than larger 

tumours. 

To develop on-chip assembled µF MBs for targeted drug delivery, we have optimised 

luciferin encapsulation into liposomes to perform proof-of-principle experiments in 

luciferase expressing cells in vitro and in tumours in vivo. In vitro testing using 

bioluminescence demonstrated relatively stable encapsulation of luciferin. Injection of 

luciferin liposomes into a luciferase expressing human CRC xenograft showed clear tumour 

specific signal. This approach will facilitate pre-clinical evaluation of MB vehicles for UStriggered 

targeted drug delivery. 

Additional Authors: 

Nicola Ingram, Elizabeth Valleley, Tony Evans 1 , Pam Jones, Alex Markham and Louise Coletta; 

Leeds Institute of Molecular Medicine; 1 Leeds Institute of Genetics, Health and Therapeutics


16 th & 17 th July 2012 



Title: Therapeutic Uses of Microbubbles: Heating, Erosion and Delivery. 

Robin Cleveland 

University of Oxford 

Abstract: The biomedical use of microbubbles was initially driven by the desire to produce 

ultrasound contrast agents for diagnostic imaging. However, microbubbles also have utility 

as a mechanical catalyst for therapeutic use ultrasound and a few applications will be 

reviewed here. In focused ultrasound surgery where HIFU is used to thermally ablate tissue 

the presence of the appropriate number of cavitation bubbles can result in significant 

increases in heating rate—resulting in shorter treatment time. A challenge is that cavitation 

nucleation is stochastic and hard to control and microbubbles can act as cavitation nuclei 

with a well characterized threshold that allows for reliable generation of cavitation bubbles. 

A second application, is non-thermal tissue fractionation, also known as histotripsy, where 

the mechanical action of cavitation bubbles can be used to erode tissue. This is a second 

case where having a well defined cavitation nucleation threshold allows for better control of 

the tissue damage and microbubbles can play that role in this application as well. Finally, 

gentle cavitation activity in the vicinity of cells, results in microstreaming that can disrupt cell 

membranes allowing for sonoporation. This has application in delivering drugs through the 

endothelium, in particular, the blood-brain barrier.


16 th & 17 th July 2012 



Title: Contrast Enhanced Ultrasound in Clinical Practice 

Jane Smith 

St James’s University Hospital 

Abstract: Contrast Enhanced Ultrasound (CEUS) has been in clinical use for the last 2 

decades. Originally used as cardiac agents and then as vascular agents, they established the 

patency of blood vessels and identified stenoses - for example in the carotid artery. First 

generation microbubble agents were relatively unstable, easily burst (even on low power 

settings) and unable to withstand repeated passage through capillary beds. 

Improvements in Doppler technology started to render contrast redundant, until it was 

discovered that deliberate microbubble destruction, in response to increased pressure, was 

able to briefly highlight liver metastases 1 - a technique known as stimulated acoustic 

emission. 

Although this technique was never a suitable tool for routine practice, it led to the 

investigation of microbubbles in detecting and characterising liver lesions, giving rise to a 

second generation of stable microbubble agents and developments in technology which 

allow operators to evaluate the contrast patterns for diagnostic purposes. 

Currently CEUS is routinely used to characterise incidental liver lesions, and also to detect 

secondary liver cancers. However its use can be translated into various other applications 

involving abdominal organs and vascular structures 2 and CEUS has become part of the 

patient pathway in many European and Canadian diagnostic centres. 

Microbubble agents have the advantage of being safe, non-nephrotoxic and easily tolerated 

by patients. CEUS has been shown to be at least as good as other imaging (for example MRI 

and CT) in characterising liver lesions and detecting metastases 3 , and it is valuable as a quick 

and accurate problem solver, often obviating the need for further diagnostic imaging. 

It’s use is slowly broadening in clinical practice to include other applications, supporting 

innovative and cost effective patient pathways. 

References: 

1.: Blomley et al: Improved Imaging of Liver Metastases with Stimulated Acoustic Emission in the Late Phase of 

Enhancement with the US Contrast Agent SH U 508A: Early Experience , Radiology, February 1999, 210, 409-416. 

2 The EFSUMB Guidelines and Recommendations on the Clinical Practice of Contrast Enhanced Ultrasound (CEUS) Update 

2011 on non-hepatic applications. 20/12/2011. http://www.efsumb.org/guidelines/guidelines01.asp 

3. Trillaud et al. Characterization of focal liver lesions with Sonovue-enhanced sonography: international multicenter-study 

in comparison to CT and MRI. World J Gastroenterol 2009; 15(30): 3748-56


16 th & 17 th July 2012


16 th & 17 th July 2012 

Number Name Title 

1 Mostafa Abdelrahman 

2 Radwa Abou-Saleh 

Poster Presentations 

Frequency dependence of flow sensitivity in Power 

Doppler and CEUS Imaging 

Developing and investigating therapeutic 

microbubbles 

3 Jennifer Bain Biomimetic synthesis of magnetic nanoparticles. 

4 Jonathan Blockley 

5 Jonathan Casey 

6 Michael Conneely 

Ultrasound measurements of known microbubble 

populations on a standard microscope stage 

Single Bubble Sub-harmonic Characterisation of Lipid 

Shelled Microbubbles: Effect of Functionalization and 

Adherence 

Computationally assisted design and experimental 

validation of a novel ‘flow-focused’ microfluidics chip 

for generating monodisperse microbubbles. 

7 Kevin Critchley Cadmium Free Quantum Dots for Biomedical Imaging 

8 Pratik Desai Ultrasound mediated microbubble generation 

9 Joe Fiabane 

10 Sevan Harput 

A high-Reynolds number microfluidic device for 

microbubble generation 

Separating the Second Harmonic Response of Tissue 

and Microbubbles using Bispectral Analysis 

11 George Heath Actin Coated Microbubbles 

12 Nicola Ingram 

13 Fairuzeta Ja'afar 

Pre-clinical evaluation of VEGFR2 as an imaging 

biomarker and targeting molecule for therapeutic 

delivery using contrast-enhanced high-frequency 

ultrasound. 

Binding Differentials of Microbubbles to Activated 

versus Non-Activated HUVECs


16 th & 17 th July 2012 

14 Tom Kokhuis Mutually interacting targeted microbubbles 

15 Klazina Kooiman Binding area of targeted microbubbles 

16 Gael Leaute 

17 Jonathan Loughran 

18 Gemma Marston 

Finite element modelling of non-spherical 

microbubble dynamics in the vicinity of a soft 

membrane 

Investigation into the Effects of Ultrasound on 

Attached Microbubbles 

Imaging the development of functional tumour 

vasculature, and the effect of combretastatin A-4 on 

tumour blood flow rate in vivo 

19 Jono McKendry Cerato Ulmin a new UCA shell material? 

20 Sally Peyman 

21 Linsey Phillips 

22 Ben Raiton 

23 Sara Shakil Qureshi 

24 Peter Smith 

25 Peter Smith 

High production rates of therapeutic microbubbles for 

targeted drug delivery. 

New Nanodroplets Increases Tissue Ablation in 

Response to High Intensity Focused Ultrasound 

Single-well acoustic tweezers using ultrasonic 

travelling waves. 

Hardware accelerated beamforming for microbubble 

detection 

Composite High Frequency, Low Frequency System for 

Pre-Clinical Microbubble Sonoporation 

Development of an Ultrasound Array Research 

Platform (UARP)


16 th & 17 th July 2012 

Poster Number: 1 

Mostafa Abdelrahman 


Title: Frequency dependence of flow sensitivity in Power Doppler and CEUS Imaging 

Abstract: 

Introduction: 

The task of detecting and measuring tumour blood flow is critical for both pre-clinical 

research and clinical diagnosis. It is well known that this is enhanced if contrast bubbles is 

used, but the optimisation of the scanning conditions continues to be somewhat 

controversial, especially for high frequency pre-clinical scanning. In addition, the role of 

Power Doppler is also unclear. 

In this study, we have used repeat scanning under a variety of conditions to compare and 

optimise sensitivity to blood flow. 

Materials and methods: 

DLD1 colorectal cancer cell xenografts were grown subcutaneously in CD-1 nude mice. The 

animals were scanned using Power Doppler ultrasound (PDUS), contrast-enhanced PDUS 

(CE-PDUS), and contrast-enhanced ultrasound (CEUS). Images were acquired in 2D and 3D 

and at 25- and 40-MHz frequencies. After imaging, mice were sacrificed, imaged with PDUS 

(control) and the xenografts were then fixed and processed for histology and 

immunohistochemistry. Images from live and dead animals were scored for colour pixel 

density and functional parameters, such as peak enhancement (PE) and perfusion rate 

(slope), were derived from CEUS time-intensity-curves (TIC). 

Results: PDUS was found to have a very poor signal to noise ratio (live animals scoring to 

dead animals scoring) due to tissue clutter and motion artifacts. However, it showed 

significant enhancement in flow detectability after the injection of bubbles. The 

enhancement was comparable for the 25- and 40-MHz transducers and was only significant 

in 2D imaging. CEUS was found to have higher detectability than CE-PDUS in 2D and 3D, and 

that was more evident at 25-MHz. At 40-MHz there was a significant correlation between 3D 

imaging and PE (R2 = 0.74 (p < 0.001) and 0.96 (p < 0.0001) for 3D PDUS and 3D CEUS 

respectively), and at 25-MHz there was significant correlation between 2D CEUS and PE (R2 = 

0.74, p < 0.0001) and between 3D CEUS and TIC slope (R2 = 0.74 (p < 0.005)) 

Conclusion: PDUS without bubbles is not reliable for the comparison of tumour vascularity 

between animals. Although 40-MHz transducer provided superior spatial depiction of the 

vascular network, the 25-MHz transducer had superior sensitivity to blood flow, especially in 

the CEUS mode.


16 th & 17 th July 2012 


Title: Developing and investigating therapeutic microbubbles 

Radwa Abou-Saleh 


Abstract: Microbubbles have been used previously as ultrasound-assisted drug delivery 

systems, as they can be targeted towards and imaged at the desired regions to enhance the 

drug therapy action. Conjugates of liposomes and gas filled microbubbles have evolved as a 

better route for drug delivery using ultrasound to rupture the microbubble and/or 

liposomes, and therefore releasing the drug at the required location. The frequency 

response of microbubbles to ultrasound excitation mainly depends on the mechanical 

properties of the shell which can be determined using atomic force microscopy (AFM). The 

addition of different components to the shell architecture can change the bubbles 

mechanical properties and therefore ultrasound response. 

Here we are preparing microbubbles with a phospholipid shell in a microfluidic device with 

different coatings. For drug delivery purposes, liposomes encapsulating Quantum Dots (Qdots), 

Fluorescein or Luciferin is attached to the bubbles and their delivery dose estimated. 

Force spectroscopy measurements with tip-less cantilevers, on microbubbles, have been 

performed to characterize the shell stiffness of a range of different shell coatings, 

specifically, Streptavidin and Q-dots. Forces ranging from 20-100 nN were applied to single 

bubbles to determine the change in stiffness with applied force. It was observed that the 

shell stiffness of phospholipid microbubbles increases linearly with increasing applied force. 


Radwa H. Abou-Saleh 1 , Sally A. Peyman 1 , Neil H.Thomson 1,2 and Stephen D.Evans 1 

1. School of Physics and Astronomy, University of leeds, LS2 9JT, UK 

2. Dept. of Oral Biology, Leeds Dental Institute, LS2 9LU, UK


16 th & 17 th July 2012 


Title: Biomimetic synthesis of magnetic nanoparticles. 

Jennifer Bain 


Abstract: Magnetotactic bacteria use an internal vesicle synthesis process for the formation 

of magnetite nanoparticles (MNPs) within the vesicle (magnetosomes). This biological 

process displays high levels of control and monodispersity with respect to MNPs. Formation 

within an internal vesicle with the aid of biomineralisation proteins allows such control to be 

realised. 

Exploitation of this process via biomimetic precipitation of MNPs within an asymmetric 

vesicle to form an artificial magnetosome is being developed, which could have applications 

in a range of biomedical and data storage nanotechnologies. The simplest route to 

magnetite MNP formation is co-precipitation. If it is possible to engineer co-precipitation 

inside an asymmetrical vesicle, forming a single magnetite crystal, it will essentially mimic 

the formation and ideally the behavior of a magnetosome. Magnetospirillum magneticum 

protein; Mms6, has been expressed and purified in previous work1. Incorporation of Mms6 

into this system should ensure MNP uniformity with regards to both the particle 

composition and consequently magnetic properties. To date three routes to co-precipitation 

have been established and optimized for the system, early results have shown encapsulation 

within an amphiphilic AB-block-copolymer vesicle to be promising. 


Jennifer Bain, Dr. Jonathan Bramble, Dr. Andrea Rawlings, Dr. Sarah Staniland


16 th & 17 th July 2012 


Jonathan Blockley 

Institution: University of Leeds 

Title: Ultrasound measurements of known microbubble populations on a standard 

microscope stage 

Abstract: Ultrasound measurements were made on lipid microbubbles in the frequency 

range 1-15 MHz. Microbubbles were produced using a microfluidic technique that gives a 

narrow dispersion of microbubble size, and some control over mean radius. 

The apparatus allows ultrasound attenuation measurements to be made on a small 

population of microbubbles while simultaneously imaging with a standard upright optical 

microscope. The microscope can be focused at a depth of choice to view the microbubbles 

as they rise, or survey the microbubbles after they have risen to the top of the chamber. 

Thus the size distribution and concentration of the microbubbles can be ascertained in situ. 

Attempts to confine the microbubbles in the observation area for prolonged testing have 

been partially successful, but the technique is highly size dependent.


16 th & 17 th July 2012 


Jonathan Casey 


Title: Single Bubble Sub-harmonic Characterisation of Lipid Shelled Microbubbles: Effect of 

Functionalization and Adherence 

Abstract: 

Introduction: The detection and utilisation of targeted microbubble (MB) ultrasound contrast agents 

for molecular imaging and targeted drug/gene delivery can be improved by making use of acoustic 

phenomena arising as a result of targeting or proximity to the target area[1]. Due to a number of 

factors; for example low MB concentration at the target site and signal masking by bubble subpopulations 

of varying size, these differences are small and difficult to deduce from standard bulk 

acoustic methods used for the characterisation of MBs. To better understand how the dynamics of 

whole populations of MBs are affected by factors such as, sub-population size, shell chemistry and 

targeting it is essential to first understand the acoustic response of single MBs. Previous work by the 

author has examined the fundamental and second harmonic response of MBs under adhesion [2]. 

The objective of this study was to examine how the sub harmonic (SH) response of MBs changes as a 

function of adhesion. SH scattering shows particular promise for MB imaging because the 

surrounding tissue does not produce any SH non-linearity. 

Methods: Single bubble acoustics was performed on in-house produced phospholipid MBs ranging in 

diameter from 1-10μm. The testing rig consisted of a pair of focussed transducers (transmit 

transducer centre frequency 3.5 MHz, receive transducer centre frequency 1 MHz) and a 40x 

microscope objective lens co-focally aligned on a capillary fibre. MBs were insonated with 2 MHz, 

10cycle, 275 KPa peak negative pressure pulses to establish the acoustic characteristics. 

To determine the effect of binding MB were examined either bound to the capillary fibre via a Biotin- 

Streptavidin bond or left unattached by the removal of one or both or the binding elements. 

Results/Discussion 

SH scattering was detected in all testing regimes. In agreement with theory the generation of SHs is 

seen to be a size dependant phenomenon, occurring predominantly when the MBs are activated 

near their resonant frequency. There was no measureable difference detected in this SH scattering 

between the adherent and free MBs at this size. SH scattering was also detected when a MB 

was driven at twice the resonant frequency. In this size region adherent MBs exhibited an increase in 

SH scattering in comparison with their unattached counterparts. Increasing ability to control the size 

distribution statistics of MBs the differences identified between adherent and free MBs could play an 

important role in their identification in targeted ultrasound diagnosis and therapy. 

MBs could play an important role in their identification in targeted ultrasound diagnosis and therapy. 

References: 

1. Dayton, P.A., Improving Technology for Molecular Imaging with Ultrasound. IEEE, 2009. 

2. Casey, J et al., Single bubble Acoustic Characterisation and Stability Measurement of Adherent 

Microbubbles, IEEE IUS-2011 


Jonathan Casey1, Charles Sennoga2, Meng-Xing Tang2, Robert Eckersley3 

1Imaging Sciences Dept, Imperial College London, United Kingdom, 2Dept of Bioengineering, Imperial 

College London, United Kingdom, 3Biomedical Engineering Dept, Kings College London, United 

Kingdom


16 th & 17 th July 2012 


Michael Conneely 

University of Dundee 

Title: Computationally assisted design and experimental validation of a novel ‘flow-focused’ 

microfluidics chip for generating monodisperse microbubbles. 

Abstract: Whilst initially developed as a diagnostic aid to improve echogenicity in ultrasound 

imaging, gas-filled lipid microbubbles are now emerging as a next generation 'theranostic' 

tool in the medical arena. Here, their therapeutic potential has now been realized through 

their unique capability to deliver molecular species such as drugs and genes by means of 

disrupting the cell membrane in response to ultrasound wave stimulus. 

The main aim for the present study was to computationally validate an in-house design for a 

novel glass microfluidic chip. The chip features a distinctive junction geometry, wherein the 

liquid and gas phases meet, and more importantly, produce stable monodisperse 

microbubbles (which may exhibit a more easily controlled, and thus clinically reliable 

response to pulsed ultrasound). The shell material in our microbubbles was composed of 

PEGylated neutral lipid mixed with cholesterol and including a gas core of either nitrogen or 

perfluorobutane. Microbubble diameters were formed within the range: 2-10μm depending 

upon the liquid and gas flow parameters. COMSOL Multiphysics was employed to create a 2- 

D simulation of the chip, and for validation, this was compared directly against our 

observational results. Figure 1 shows the bubble formation process as observed with a high 

speed camera (above) and as modeled in COMSOL (below). Future parameterized studies 

implemented via COMSOL, will allow us to ascertain the factors that will inform next 

generation chip-design, as well as the optimum operational parameters. 


Michael Conneely* 1 , Vikas Hegde 2 , Hans Rolfsnes 1 , Adrian Mason 2 , Charlie Main 1 , Frances JD Smith 2 , 

WH Irwin McLean 2 & Paul A Campbell 1,2 

1 Carnegie Physics Laboratory, University of Dundee, Dundee DD1 4HN. Scotland UK 

2 Division of Molecular Medicine, University of Dundee, Dundee DD1 4HN. Scotland UK 

Figure 1. Comparison between experimental (top) and numerical model (below) results.


16 th & 17 th July 2012 


Title: Cadmium Free Quantum Dots for Biomedical Imaging 

Kevin Critchley 


Abstract: Quantum dots (QDs) have been predicted to make a big impact in the 

development of biomedical imaging [1] and significant progress has been made with QDs 

being used for targeted imaging in small animals [2]. However, the majority of biomedical 

QD studies to date have utilised cadmium based QDs and this raises important questions 

regarding toxicity [3]. In order to develop a QD system that could be potentially introduced 

into a clinical environment the semiconductor must be cadmium free. In this study, the 

photoluminescence (PL) quantum efficiency of heavy metal free CuInS2/ZnS quantum dots in 

aqueous solution is investigated. CuInS2 possesses a narrow band gap enabling biologically 

important near infra-red emission to be easily achieved, and complete surface passivation 

with ZnS results in high quantum efficiency. We consider the PL response to changes in pH, 

temperature and buffer in order to quantify their behaviour in the biological environment. 

Importantly we also examine the biocompatibility and long-term photostability, providing 

useful information for the application of these promising quantum dots in biological assays. 

The role that surface chemistry has in providing both the stability in solution and the 

potential for bioconjugation required for biological applications proves to be significant [4]. 

References 

[1] I. Medintz, H. Uyeda, E. Goldman et al. Nature Materials, 4, 6 (2009), 435-446 

[2] A. Hoshino, K. Hanaki, K. Suzuki et al. Biochem. and Biophys. Res. Comm. 314, 1 (2004) 

46-53 

[3] B. Rzigalinski, J. Strobl, Toxicology and Applied Pharmacology, 238 (2009), 280-288 

[4] J. Weng, J. Ren, Current Medicinal Chemistry, 13, 8 (2006), 897-909 


Matt Booth and Kevin Critchley 

School of Physics and Astronomy, University of Leeds, UK


16 th & 17 th July 2012 


Title: Ultrasound mediated microbubble generation 

Pratik Desai 

University of Sheffield 

Abstract: Ultrasound mediated drug delivery via microbubbles is a technique that could 

potentially revolutionise the treatment of tumours and is currently under study in many 

research laboratories. Before being used as drug delivery vectors, however, microbubbles 

need to be manufactured: a monodisperse distribution centred at a diameter smaller than 6- 

8 µm is the desired target (to account for the capillary system). In this paper, the authors 

investigate the use of an acoustic modulation to pilot the size of bubbles produced by a 

pressure-controlled microfluidic actuator. In this study, the authors added a bespoke diffuser 

to the pre-conditioning actuator, so as to induce the production of smaller bubbles & further 

increase the throughput of the system. A resonating cavity was used to induce vibration of 

the diffuser at a designed frequency. As shown in Figure 1, the performance of the system 

changed as the acoustic oscillator approached resonance: the throughput of the microfluidic 

actuator was increased manifold and generated a narrower bubble size distribution, coupled 

with a decrease in bubble size (75 % between 3-4 μm) The design at NPL of an in house 

detection system (Figure 2), capable of detecting bubbles up to a size of 3 μm, was an 

important aspect of the project. Inverse problems posed by the Fredholm integral, necessary 

to garner a size distribution from the raw data, were solved numerically. Use of protein /lipid 

coating & PFC core will further stabilise the bubble & reduce size. If successful, the concept 

could lead to a substantial reduction in the cost of the currently expensive conventional 

encapsulated microbubbles for medical imaging, even in other microfluidic-based 

manufacturing techniques. 

Figure 1 Bubble Size Distribution at different frequencies Figure 2 Rig 


P.D. Desai* 1 , G. Memoli 2 , D. Kuvshinov 1 and W. Zimmerman 1 

Affiliation: 1 Department of Chemical & Biological Engineering, University of Sheffield; 2 Acoustics Group, 

National Physical Laboratory


16 th & 17 th July 2012 


Joe Fiabane 

Robert Gordon University, Aberdeen 

Title: A high-Reynolds number microfluidic device for microbubble generation 

Abstract: A key requirement of the generation of microbubbles for use in drug delivery is 

narrow size distribution and close control over size, as this is a dominant factor governing 

ultrasound response. Microfluidic devices offer superior performance in this regard, 

compared with conventional production methods such as sonication and mechanical 

agitation. However previously reported devices require very small internal geometry, 

necessitating high operating pressures and resulting in frequent blockages. Hence it is 

desirable to develop a device which produces microbubbles much smaller than its internal 

channel widths. The best device thus far reported produces microbubbles one tenth of the 

minimum channel width. We present a device capable of producing microbubbles as small as 

2.5 μm via a minimum channel diameter of 100 μm. Pressurised air surrounds an annular 

gas-in-liquid jet passing through a 100 μm orifice. The jet diameter, and hence the resulting 

microbubble diameter, can be controlled simply by varying the surrounding air pressure. 

Furthermore, the device operates in a novel high-Reynolds number regime, which would 

typically be expected to yield highly polydisperse results. However this device is able to 

maintain a relatively narrow size distribution with polydispersity index 16% at optimal 

performance. Operating regimes are described and size distribution data are presented. 

Studies have also been carried out to investigate the effect of varying core gas and shell 

composition on the effect of microbubble stability. 


Joe Fiabane*, Ritu Malik, Prem Nagappanpillai, Paul Prentice, Ketan Pancholi


16 th & 17 th July 2012 


Sevan Harput 


Title: Separating the Second Harmonic Response of Tissue and Microbubbles using Bispectral 

Analysis 

Abstract: The second harmonic generation in medical ultrasound is either caused by tissue or 

ultrasound contrast agents (UCAs). The conventional signal processing techniques cannot separate 

the harmonic response from microbubbles and tissue. The second order spectral analysis, commonly 

known as the frequency analysis, is the most common way of evaluating the microbubble response. 

Although frequency analysis can estimate the power spectrum effectively, it suppresses the phase 

relation between the frequency components. In this study, third order (bispectral) analysis is used to 

evaluate the microbubble response and separate the second harmonic generated by tissue and 

microbubbles via the phase coupling between fundamental and harmonic components. 

To verify the validity of the proposed technique, an ultrasound phantom was created with tissue 

mimicking material (TMM) and a chamber for UCAs. A standard medical probe was used to scan the 

phantom using a 96-channel Ultrasound Array Research Platform (UARP). An LFM chirp sweeping 

between 3-4 MHz was used for excitation and the second harmonic was received by the same probe 

at 6-8 MHz. It was observed in the measurements that CTR can drop below 0 dB for low UCA 

concentrations as shown in the figure, where it is impossible to distinguish between TMM and 

microbubbles. However, bispectral analysis showed phase coherence between fundamental and 

second harmonic for TMM as high as 83%. No significant correlation between the second harmonic 

and fundamental components was observed for microbubbles, where the phase coherence value 

was less than 10%. 

Results show that higher-order spectral analysis allows the separation of the harmonics generated by 

microbubbles and tissue. Unlike other tissue harmonic suppression techniques based on multipleexcitations, 

bicoherence is not susceptible to motion artefacts since movement does not affect the 

phase relation between fundamental and harmonic components. 


Sevan Harput, James McLaughlan, Peter R. Smith, 

David M. J. Cowell, Stephen D. Evans a 

and Steven Freear 

Ultrasound Group, School of Electronic and 

Electrical Engineering University of Leeds, Leeds, UK.; 

a School of Physics & Astronomy, University of Leeds, 

Leeds, UK.


16 th & 17 th July 2012 


Title: Actin Coated Microbubbles 

George Heath 


Abstract: The protein actin forms long semi-flexible polymers known as microfilaments 

found as a supporting cortex in all eukaryotic cells with the function of supporting the cell 

membrane and shape. We currently utilize this to create novel biomimetic microbubble 

coatings which will potentially allow subharmonic ultrasound imaging by creating nonlinear 

radial oscillations. We use cationic lipids to polymerise actin at the lipid-liquid interface 

creating a lipid microbubble with a monolayer coating of actin filaments. Actin-microbubbles 

were characterised with florescence microscopy, AFM force spectroscopy and bubble 

dissolution. These techniques show the actin shell increases bubble stiffness, resistance to 

gas permeation and elasticity when compared to lipid alone. We plan to further this coating 

by adding actin binding proteins with crosslinks, branches and capping proteins, in an aim to 

create a network with tension.


16 th & 17 th July 2012 


Nicola Ingram 


Title: Pre-clinical evaluation of VEGFR2 as an imaging biomarker and targeting molecule for 

therapeutic delivery using contrast-enhanced high-frequency ultrasound. 

Abstract: We are developing novel ways to image functional vasculature in vivo for assessing 

tumour development and response to therapeutics as well as delivering tumour targeted 

therapeutics for treatment of colorectal cancer (CRC). VEGFR2 has been described as a 

marker of angiogenesis but its expression and distribution in colorectal xenograft tumour 

development has not been determined. We have therefore used contrast enhanced high 

frequency ultrasound (CE HFUS) for dynamic imaging of VEGFR2 in a longitudinal study of 

SW480 CRC xenografts. 

We used the VisualSonics Vevo 770 micro-US system to evaluate bolus injection 

characteristics to show functional vessels within the tumour and binding of MBs to tumour 

vasculature in 2D. In addition, we validated VEGFR2-targeted binding of Micromarker 

microbubbles and microfluidics-generated microbubbles in vitro under flowing conditions. 

Immunohistochemistry on SW480 xenografts showed that VEGFR2 was indeed present in 

tumour vessels, in line with human studies. There was a peak in CD31+ vessels that also 

expressed VEGFR2 at approximately 50mm3 in size. Expression then decreased as the 

tumours increased in volume. Both Micromarker and microfluidics microbubbles specifically 

bound VEGFR2+ve cells under flowing conditions. VEGFR2-targeted MB injection and 

subsequent destruction of bound MBs showed a greater binding of microbubbles in smaller 

tumours. 

VEGFR2 expression was upregulated in small, developing CRC xenografts and can be 

therefore used to image vasculature and target microbubbles in pre-clinical models of CRC.


16 th & 17 th July 2012 


Fairuzeta Ja'afar 


Title: Binding Differentials of Microbubbles to Activated versus Non-Activated HUVECs 

Abstract: There is growing evidence to show that late-phase contrast-enhanced ultrasound 

imaging (LP-CEUSI) of the carotid using non-targeted commercial microbubbles (MBs) 

reflects biological features of inflammation.1 Whereas a number of potential initiators and 

mechanism(s) have been proposed as suitable explanations for this non-targeted MB 

retention in the vasculature, much uncertainty remains about how these vascular cues 

influence MB adhesion. In this study, we evaluated the binding differentials of various 

commercial MBs (SonoVue, Optison and Definity/Luminity) and the experimental 

agent BR38, with models of inflamed (activated) and normal (non-activated) vasculature in 

vitro. We employed human umbilical vein endothelial cells (HUVECs) and observed MB 

binding using bright-field microscopy under physiological flow conditions. We then examined 

the role of MB surface charge on HUVECs/MBs binding affinities using a combination of laser 

Doppler velocimetry (LDV) and phase analysis light scattering (PALS) on a Zetasizer Nano Z 

(Malvern Instruments). We found a ~3-fold and ~1-fold increase in the number densities of 

Optison and SonoVue MBs adherent respectively, on activated versus non-activated 

HUVECs (p


16 th & 17 th July 2012 


Title: Mutually interacting targeted microbubbles 

Tom Kokhuis 

Erasmus MC 

Abstract: Ultrasound application can rupture the adhesion of targeted microbubbles (MBs) due to a mutual 

interaction between the bubbles called secondary Bjerknes force (SBF) 1,2 . At lower pressures, targeted MBs 

were observed to move up to several hundreds of nanometers towards each other during ultrasound 

application, but recoiled back again to their initial position within 100 �s after the ultrasound was turned off 2 . 

Interestingly, the MBs remained spherical throughout the experiments. In this study we investigated the origin 

of this elastic restoring force in more detail using simultaneous top and side view high-speed imaging. 

Homemade biotinylated MBs were prepared by sonication as described by Klibanov et al 3 and targeted to the 

topside of an avidin-coated polystyrene capillary. Isolated pairs of MBs were insonified with 20 cycles at 2.25 

MHz (P_ = 0 – 200 kPa). Oscillatory and translationary dynamics were imaged with two orthogonally placed 

lenses. Images were superimposed and relayed to the Brandaris128 camera 4 . A typical example of the effect of 

SBF on the position of targeted MBs (in top view) is shown in panel A. The attractive SBF caused each MB to 

move by about 600 nm towards the other MB. In top view the MBs did not exhibit significant deformation, 

confirming earlier observations. However, side view imaging revealed the attractive SBF causes a MB to 

elongate in the direction of the other MB, thereby tending towards a prolate shape. This effect is illustrated in 

panel B for bubble 2. The aspect ratio of the MB as seen from the top (red dots) is approximately constant and 

near unity throughout the experiment. However, the aspect ratio from the side (blue dots) increases by about 

20% from 1.1 to 1.3. Panel C (top view) and panel D (side view) show the corresponding images of the MBs at t 

= 0 �s and t = 11.26 �s (i.e. last frame). 

These results show that the attractive force between pulsating targeted MBs is opposed by a restoring force 

due to bubble deformation, which is driving the recoil after the US is turned off. The recoil could be modeled 

with a simple mass-spring system, yielding a value for the spring stiffness k of the order 10 -3 N/m. Moreover, 

the translational dynamics of interacting MBs was predicted by a hydrodynamic point particle model, including 

a value of the spring stiffness k of the very same order as derived experimentally from the recoiling curves. 

Understanding the effect of ultrasound application on targeted MBs is crucial for further advances in the realm 

of molecular imaging. 

References: 

[1] Schmidt et al., J Control. Release, 131 (1), 2008 

[2] Kokhuis et al., Proc. IEEE Ultrason. Symp., Orlando, 2011 

[3] Klibanov et al., Invest. Radiol., 39 (3), 2004 

[4] Chin et al., Rev Sci Instr, 74 (12), 2003 


T.J.A. Kokhuis 1,7 * , B.A. Naaijkens 2,7 , L.J.M. Juffermans 3,7 , O. Kamp 4,7 , M. Versluis 5,6 and N. de Jong 1,7 

1 Biomedical Engineering, Thorax Center, Erasmus MC, Rotterdam, the Netherlands 

2 Department of Pathology, VU Medical Center, Amsterdam, the Netherlands 

3 Department of Physiology, VU Medical Center, Amsterdam, the Netherlands 

4 Department of Cardiology, VU University Medical Center, Amsterdam, the Netherlands 

5 Physics of Fluids Group, University of Twente, Enschede, the Netherlands and MIRA Institute of 

Biomedical Technology and Technical Medicine, University of Twente, Enschede, the Netherlands 

7 Interuniversity Cardiology Institute of the Netherlands, Utrecht, the Netherlands


16 th & 17 th July 2012 


Title: Binding area of targeted microbubbles 

Klazina Kooiman 

Erasmus MC 

Abstract: For molecular imaging using ultrasound contrast agents, targeted microbubbles (tMB) are designed 

with specific ligands linked to the coated shell 1 . Research is ongoing to determine the binding force of tMB and 

to distinguish bound from unbound tMB using ultrasound 2,3 . For this, the actual surface of the tMB that binds 

to a pathology is important. This study focuses on determining the binding area of bound tMB by fluorescence 

microscopy. 

Lipid-coated MBs (3-7 µm in diameter; composition in mol%: DPPC or DSPC 59.1; PEG-40 stearate 35.7; DSPE- 

PEG(2000) 4.1; DSPE-PEG(2000)-biotin 0.8) with a C4F10 gas core were made by sonication 4 . Binding area of 

tMB was studied by adding the lipid dye DiD to the MB before sonication and coating an Opticell membrane 

with fluorescent streptavidin Oregon Green 488 (5 µg/ml). High-resolution images were recorded using 4Pi 

microscopy 5 . The overlap of the red and green fluorescence was calculated using Fiji software on basis of 

intensity threshold. 

The binding area of bound tMB was found to be 11 ± 4% of the total MB surface for tMB with DPPC as the main 

lipid (n=24) and 6 ± 4% for tMB with DSPC as the main lipid (n=22). Examples are given in the Figure. The 

average binding area was smaller for the DSPC tMB and can be explained by the heterogeneous distribution of 

the ligand throughout the MB coating 6 . These findings can be used to improve the binding of tMB and for the 

ongoing research to distinguish bound from unbound tMB. 

References: 1 Lindner et al, Nat Rev Cardiol 6:475, 2009; 2 Dayton et al, Mol Imaging 3:125, 2004; 3 

Zhao et al J Acoust Soc Am 120:EL63, 2006; 4 Klibanov et al, Invest Radiol 39:187, 2004; 5 Hell et al, J 

Microsc-Oxf 187:1, 1997; 6 Kooiman et al, IEEE Proceedings 2010. 

Klazina Kooiman *a , Tom J.A. Kokhuis a,b , Ilya Skachkov a , Johan G. Bosch a , Antonius F.W. van der Steen 

a,b , Wiggert A. van Cappellen c , Nico de Jong a,b 


a Dept. of Biomedical Engineering, Thoraxcenter, Erasmus MC, Rotterdam, the Netherlands 

b Interuniversity Cardiology Institute of the Netherlands, Utrecht, the Netherlands 

c Dept. of Reproduction and Development, Erasmus MC, Rotterdam, the Netherlands 

Figure. Targeted microbubbles (red fluorescent) bound to a streptavidin-coated surface 

(green fluorescent) recorded by 4Pi microscopy. A) microbubble with DPPC as main 

lipid (4.7 µm in diameter); B) microbubble with DSPC as main lipid (4.5 µm in 

diameter). 

This research is supported by the 

Center for Translational Molecular 

Medicine and the Dutch Heart 

Foundation (PARISk).


16 th & 17 th July 2012 


Gael Leaute 


Title: Finite element modelling of non-spherical microbubble dynamics in the vicinity of a 

soft membrane 

Abstract: A combination of microbubbles and ultrasonic insonation can increase the 

permeability of a cell membrane to increase uptake of a therapeutic drug. Previous studies 

have shown that non-spherical deformation can occur near a boundary, often forming jets 

that may create localised cell membrane damage.1 In this study, a visco-elastic finite 

element model for simulating the response of a phospholipid encapsulated microbubble to 

ultrasound in the vicinity of a soft membrane was implemented. The model was used to 

study the non-spherical deformations at high insonation pressures and investigate the 

factors which affect the likelihood of jetting. 

Transient response of a microbubble was simulated with Comsol Multiphysics(TM) Finite 

Element Analysis software. Microbubbles with an initial radii of R0 = 1-5 µm and a 2 nm thick 

lipid coating, situated from 100nm to R0 a soft membrane were considered. The ratio ξ=d/R0 

and the amplitude of the ultrasound field were considered to study the likelihood of jetting, 

where d is the shortest distance between the cell membrane and the microbubble wall. 

The distance d at which jetting occurs was found dependent on the microbubble radius and 

the peak negative pressure. For a pressure of 500 kPa and ξ ≤ 0.4 jetting was present and 

increased in magnitude as d was reduced. However, for ξ > 0.4 non-spherical shell 

deformations were present, but no jetting was observed. For ξ < 0.08 a minimum Pa of 200 

kPa was sufficient to establish jetting. The likelihood of jetting was found dependent on ξ 

and as d decreased the pressure required for jetting also decreased. This could suggest that 

targeted microbubbles bound to a cell are likely to result in jetting at lower pressures than 

unbound microbubbles. 

References: 

P. Prentice et al. Nature Physics, 1(2):107–110, 2005. 


Gaël Léauté*, David M. J. Cowell, James McLaughlan, Sevan Harput and Steven Freear


16 th & 17 th July 2012 


Title: Investigation into the Effects of Ultrasound on Attached Microbubbles 

Jonathan Loughran 


Abstract: Microbubble contrast agents have shown great potential in imaging and 

quantifying blood flow and tissue perfusion. Recent advance of targeted microbubbles have 

made it possible to detect and imaging the distribution of specific molecules of interest 

expressed on endothelial walls in vivo using ultrasound. Previous studies have been focused 

on developing microbubbles with improved targeting efficiency, acoustic techniques to 

facilitate the attachment of such bubbles and better detection method, little attention has 

been paid to study what happens to the microbubbles once they are attached. In this study 

the aim is to study the effects of ultrasound exposure to attached microbubbles. Biotin 

coated microbubbles were flown through a phantom coated with streptavidin. Optical 

microscopy was used to observe the attached microbubbles in the flow phantom under 

ultrasound exposure. The bubble size and position were recorded during the exposure of 

ultrasound. It was found that even at relatively low acoustic pressure a significant amount of 

bubbles detach from the wall and flow away, some of them shrink at the same time. The 

number of bubble detachment increases with acoustic pressure. The different forces acting 

on the bubbles during the exposure was analysed to offer some insights into the detachment 

mechanisms. 


Jonathan Loughran 1 , Charles Sennoga 1 , Robert Eckersley 2 , Meng-Xing Tang 1 * 

1 Department of Bioengineering, Imperial College London, London SW7 2AZ, UK 

2 Biomedical Engineering Department, King's College London, London. SE1 7EH, UK


16 th & 17 th July 2012 


Title: Imaging the development of functional tumour vasculature, and the effect of 

combretastatin A-4 on tumour blood flow rate in vivo 

Gemma Marston 


Abstract: 

Contrast enhanced high-frequency ultrasound (CE HFUS) enables longitudinal studies in 

mouse models and can be used to investigate both qualitative and quantitative changes in 

tumour vasculature, including tumour blood flow and perfusion. We have developed CE 

HFUS protocols with the VisualSonics Vevo770 in order to characterise the development of 

functional tumour vasculature in vivo in a murine xenograft model of colorectal cancer over 

35 days of tumour growth, and have undertaken a pilot study to image and quantify the 

effect of combretastatin A-4 (CA4) on tumour blood flow kinetics. 

Time intensity curves were generated post-acquisition and used to determine perfusion 

kinetics and vascular data. Tumour vascular space and tumour volume was calculated from 

3D CE HFUS images. This showed that the percentage tumour vascular space decreased 

significantly as the tumour volume increased. Furthermore, smaller tumours had faster rates 

of blood flow than larger tumours and smaller tumours had significantly higher maximum 

levels of vascular perfusion than larger tumours. Post treatment of CA-4 showed that the 

blood flow rate within tumours significantly decreased, with maximum intensity persistence 

showing a significant decrease in total blood flow through the tumour. 

This study shows that CE HFUS is a powerful tool for analysis of tumour vascular 

development in vivo and provides a relatively non-invasive method to assess development of 

anti-angiogenic and vascular disruptive therapies, and response to treatment in pre-clinical 

trials.


16 th & 17 th July 2012 


Title: Cerato Ulmin a new UCA shell material? 

Jono McKendry 


Abstract: The hydrophobin cerato ulmin (CU) has been used as a stabilising coating for 

bubbles on the micrometer length scale. CU coated bubbles are shown to display greater 

stability than commonly used contrast agent coating lipids, with sample lifetimes greater 

than 10 days. Using AFM force spectroscopy the mechanical properties of the cerato ulmin 

coated bubbles have been investigated. Using tipless AFM cantilevers to compress the 

bubbles their stiffness has been quantified (254 mN/m for a bubble of radius ~ 9μm) and 

dynamic visco-elastic measurements have been performed. Furthermore, methods of 

production of spherical and linear bubbles are described with discussion of the potential of 

non-spherical microbubbles as ultrasound contrast agents. 


Dr J. E. McKendry 1 , Dr C. Grant 2 , Prof P. S. Russo 3 , Prof S. D. Evans 1 

1. School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT 

2. Polymer IRC, University of Bradford, Bradford, BD7 1AL 

3. Department of Chemistry & Macromolecular Studies Group, Louisiana State University, 

Baton Rouge, Louisiana 70803-1804


16 th & 17 th July 2012 


Title: High production rates of therapeutic microbubbles for targeted drug delivery. 

Sally Peyman 


Abstract: Micron sized, lipid stabilized bubbles of gas are of interest as contrast agents for ultrasound 

(US) imaging and increasingly as vehicles for targeted drug delivery. Microfluidics provides a 

reproducible means for microbubble production and surface functionalisation using flow-focussing 

microfluidic devices that combine streams of gas and liquid through a nozzle a few microns wide and 

then subjecting the two phases to a downstream pressure drop. While microfluidics has successfully 

demonstrated the generation of monodisperse bubble populations, these approaches inherently 

produce low bubble counts. We introduce a new micro-spray flow regime that generates consistently 

high bubble concentrations, in the order of 10 10 bubbles mL -1 , that are more clinically relevant 

compared to traditional monodisperse bubble populations. The technique is shown to be easy to 

implement and highly reproducible. By using multiplexed chip arrays containing up to 4 bubble 

devices, the time taken to produce one millilitre of sample was ~10 minutes. Further, we also 

demonstrate that it is possible to attach liposomes, loaded with quantum dots or fluorescein and 

form microbubbles containing an internal layer of oil for the delivery of hydrophobic drugs, in a 

single step during MBs formation. 


Sally A. Peyman, Radwa Abou-Saleh, Stephen D. Evans. 

School of Physics and Astronomy, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT. UK.


16 th & 17 th July 2012 


Linsey Phillips 

University of North Carolina 

Title: New Nanodroplets Increases Tissue Ablation in Response to High Intensity Focused 

Ultrasound 

Abstract: Introduction: We are investigating a new low-boiling point perfluorocarbon emulsion 

composed of decafluorobutane (b.p.=2°C) and dodecafluoropentane (b.p= 30°C) for enhanced 

thermal delivery during high intensity focused ultrasound (HIFU) ablation. Microbubbles (MBs) are 

known to enhance thermal delivery, however they can shield energy deposition to deeper tissues, 

and are too large to extravasate. Our nanodroplets (NDs) are 240 +/- 65 nm in diameter, small 

enough to extravasate into tumor tissue, and are inherently more sensitive to ultrasound due to their 

low boiling point. We hypothesized that these NDs would increase HIFU ablation lesions in tissuemimicking 

phantoms compared to agent-free controls. Materials and Methods: A range of 

concentrations of MBs or NDs were added to individual albumin-acrylamide tissue-mimicking 

phantoms. A focused 1 MHz array applied continuous wave HIFU at 4 MPa for 20 seconds to 

phantoms maintained at 37°C. Temperature was monitored at the acoustic focus within the 

phantoms using a needle-type thermocouple probe. Results and Discussion: The addition of NDs or 

MBs significantly increased the size of lesions in the phantoms in response to HIFU. Lesion size was 

highly dose dependent (see Fig. 1A). The temperature rose faster in phantoms containing NDs 

compared to those containing MBs or no agents. Lesion formation occurred at the surface of 

phantoms containing MBs, wherein these MBs shielded deep lesion formation (Fig. 1B). ND lesions 

formed around the acoustic focus (Fig. 1B). Conclusion: This study demonstrates that our new 

nanodroplets containing a low boiling point PFC enhance HIFU ablation. Furthermore these agents 

induce targeted lesion formation at the site of interest while avoiding non-targeted heating. 

Additional authors: Linsey C. Phillips*, Connor Puett, Paul S. Sheeran, Paul A. Dayton 

Department of Biomedical Engineering, University of North Carolina at Chapel Hill, USA 

Figure 1 A) HIFU induced lesion volumes (mean ± S.D., n ≥ 3) in albumin-acrylamide phantoms containing 

microbubbles (MB) or nanodroplets (ND) as a function of concentration and B) corresponding images of each type of 

lesion.


16 th & 17 th July 2012 


Title: Single-well acoustic tweezers using ultrasonic travelling waves. 

Ben Raiton 


Abstract: 

SAW devices can be used in bioscience applications to manipulate micrometre-scale particles. 

“Wells” are expected to form every half wavelength within a 1D pattern and √2/2 times the 

wavelength within a 2D pattern. A well of 10s of μm can be obtained by creating a standing wave 

with a frequency of a few MHz. However, this also means that a high number of wells will form 

within a working region of a few millimetres. Two apertures emitting acoustic travelling waves with 

opposite phase results in the generation of a pressure null at the focal depth. This way, a single well 

can be obtained over a few millimetres. As is the case for the standing waves, increasing the pressure 

level will apply a stronger radiation force on the trapped particles and form a narrower well. Then, by 

translating the array or even steering the beams the trapped particles can be manipulated. 

Lipid shelled microbubbles with a size distribution of 1-10 μm are flown through a 200 μm cellulose 

tube at a rate of 7ml/hr. A linear array is set to emit 5 MHz 50 cycle tone bursts at a repetition 

frequency of 30 kHz. The whole array aperture is focused, at 30 mm, onto the tube. Although each 

element emits the same tone burst, both halves of the aperture are out of phase by 180°. Once 

sufficient microbubbles have accumulated the flow is stopped. The array is then translated along the 

tube axis to displace the trapped cluster across the microscope viewing window which has a width of 

1.2 mm. 

A concentration of 100x10 6 microbubbles /ml were flown until enough microbubbles were 

accumulated to form a cluster of 50 μm in diameter. Whilst the tube remained insonified, the flow 

was stopped. The trapped cluster was then translated within the viewing window at up to 1mm/s. 

After over 60 sec of manipulation there was no visible change in the cluster diameter. A single 

microbubble aggregation was successfully formed over the entire viewing window. The cluster could 

then be translated to any desired location along the tube. The impact on the microbubble lifetime 

was limited as they were constantly held at the pressure null. Using a 1D setup, a cluster could be 

translated over 10s of mm as long as it is restricted to a narrow channel. The same technique could 

be extended to a 2D setup so that a cluster could be freely translated across a 2D plane such as a cell 

layer.


16 th & 17 th July 2012 


Title: Hardware accelerated beamforming for microbubble detection 

Sara Shakil Qureshi 

University of Sciences and Technology, Pakistan 

Abstract: A flexible, real-time digital beamformer implementation is described for 

microbubble detection. The architecture enables acquisition of both beamformed and prebeamformed 

data of up to 96 channels from a single transmit event, and is suitable for a 

range of imaging methods such as linear array imaging, phased array imaging and synthetic 

aperture imaging. 

The Ultrasound Array Research Platform (UARP) receives ultrasound data from 96 parallel 

channels to an Altera Stratix III FPGA after 12 bit Analogue to Digital conversion at 50 MHz. 

Samples are adjusted to remove any DC Offset error caused by the Analogue Front End. 

Samples are then delayed and weighted according to delay profiles and apodization 

coefficients respectively. Each adjusted, delayed and weighted output stream in the 

aperture is then added in a pipelined adder. A clipping circuit ensures that beamformed data 

outside of a 16 bit output range is captured and does not appear as false data. All 

parameters including apodization coefficients, aperture size and output range select are 

controllable at run-time. The beamformed output is saved into on-chip memory of 4096 

samples depth, alongside the corresponding pre-beamformed data. The user has the option 

of transferring either or both forms of data to the host PC, thus offering greatest access to 

the received ultrasound data. The architecture also permits fast acquisition of microbubble 

sequences by storing multiple beamformed scanlines in memory. 


Sara S. Qureshi 12 *, Peter R. Smith 1 , Dr David M. J. Cowell 1 , Dr Kashif M. Rajpoot 2 , and Dr Steven 

Freear 1 

1 Ultrasound Group, School of Electronic and Electrical Engineering, University of Leeds, United 

Kingdom. 2 School of Electrical Engineering and Computer Science, National University of Sciences and 

Technology, Pakistan. 

Received 

Echo 

Data 

Parameter 

Loading 

Block 

Analog 

Front 

End 

DC 

Offset 

Adjust 

Block 

Delay Loading 

Apodization 

Loading 

Fine 

Delay 

Block 

Coarse 

Delay 

Block 

Software Interface (MATLAB) 

Beamformed Data Path 

Pre-Beamformed Data Path 

USB Link 

NIOS PROCESSOR 

Apodization 

Block 

Summing 

Block 

Field Programmable Gate Array (FPGA) – Stratix III 

Ultrasound Array Research Platform (UARP) 

Range 

Selection 

Block 

Data 

Read 

Block 

Pre-Beamformed 

Data 

AND 

Beamformed Data 

Memory


16 th & 17 th July 2012 


Peter Smith 


Title: Composite High Frequency, Low Frequency System for Pre-Clinical Microbubble 

Sonoporation 

Abstract: Ultrasound contrast agents or microbubbles are primarily used clinically to 

increase vasculature delineation or perform cardiac perfusion measurements. Research has 

shown a potential secondary function of microbubbles as a drug or gene delivery method for 

therapeutic applications. This secondary function relies on the microbubble oscillating under 

an applied ultrasound field. Oscillations of microbubbles in close proximity to cell 

membranes cause them to rupture temporarily - a technique known as ‘Sonoporation’. This 

technique has shown potential in enabling more targeted therapy by delivering genes or 

drug loaded microbubbles to tumour sites. 

Pre-clinical in vivo studies are the first step in evaluating the efficacy of microbubble 

drug/gene delivery. The use of small animal models for these studies has a number of 

advantages, but requires high frequency ultrasound systems to achieve satisfactory image 

quality. High frequency ultrasound may provide high resolution images, but is inefficient for 

driving microbubble oscillations. Current trends therefore seek to combine high frequency 

imaging with low frequency excitation of microbubbles. This provides best trade-off 

between image quality and Sonoporation efficiency. 

This work reports on progress of a two part study, investigating: 1) the optimum low 

frequency ultrasound parameters for microbubble destruction, and 2) using these optimised 

parameters in In vivo mouse trials with liposome-loaded microbubbles. Results are obtained 

using a high frequency (40 MHz) pre-clinical imaging system (VisualSonics Vevo 770) in 

conjunction with a custom single channel, low frequency (0.1 – 10 MHz) system. 


Peter R. Smith* 1 , Dr James R. McLaughlan 1 , Dr David M. J. Cowell 1 , Dr Nicola Ingram 2 and Dr. Louise 

Coletta 2 

1 Ultrasound Group, School of Electronic and Electrical Engineering, University of Leeds, United 

Kingdom. 2 Leeds Institute of Molecular Medicine, Wellcome Trust Brenner Building, St James’s 

University Hospital, Leeds, United Kingdom.


16 th & 17 th July 2012 


Title: Development of an Ultrasound Array Research Platform (UARP) 

Peter Smith 


Abstract: Commercial ultrasound systems enable a medical professional to perform quick 

and accurate diagnosis of medical conditions, assist in surgical procedures, or apply 

treatment. These systems provide a user with the optimal apparatus to perform specific, 

well-defined tasks in a clinical setting, but are often highly constrained or inflexible. 

In a research environment, non-conventional operation of components in the ultrasound 

system is often required. Techniques such as pulse inversion, power modulation and 

harmonic imaging have previously demonstrated non-conventional transmitter sequencing, 

signal acquisition and receive processing. A lack of system control may therefore be a 

significant barrier to research when using a clinical system. Moreover, implementation of a 

new research method may require software development and/or hardware modifications, 

where information or support from the manufacturer is required. Furthermore, the 

information needed to evaluate the new research method may not be available from the 

commercial system, or available in the correct format. For example, the output of 

commercial systems is often a set of high-definition processed images showing relative pixel 

intensity which is often an unsuitable output for researchers. Access to pre-beamformed RF 

data lets you analyze the received signal both qualitatively and quantitatively, which 

improves the diagnostic ability of the system and permits further study of results. 

In the research environment a custom flexible platform provides the user with full system 

control and offers distinct advantages over a commercial clinical system. This work reports 

on the continual development of a custom Ultrasound Array Research Platform (UARP), 

developed to offer the flexibility required for a research context. An overview of the 

platform is provided, with examples of non-conventional operation given, particularly with 

respect to ultrasound-microbubble interactions. Future developments and applications are 

also presented. 


Peter R. Smith*, Dr David M. J. Cowell, Dr James R. McLaughlan, Sevan Harput, Benjamin Raiton, 

Olaoluwa Olagunju, Sara Shakil Qureshi, Charlie Winckler and Dr Steven Freear 

Ultrasound Group, School of Electronic and Electrical Engineering, University of Leeds, United 

Kingdom


16 th & 17 th July 2012 

Delegate List 

Name Email Affiliation 

Mostafa Abdelrahman mtp5ma@leeds.ac.uk University of Leeds 

Radwa Abou-Saleh R.H.Saleh@leeds.ac.uk University of Leeds 

Mohamed Ashawesh ashawish77@yahoo.co.uk University of Sheffield 

Jennifer Bain pmjb@leeds.ac.uk University of Leeds 

Jonathan Blockley pyjb@leeds.ac.uk University of Leeds 

Ayache Bouakaz ayache.bouakaz@univ-tours.fr Université François Rabelais, 

Tours 

Richard Bushby R.J.Bushby@leeds.ac.uk University of Leeds 

Jonathan Casey jonathan.casey08@imperial.ac.uk Imperial College London 

Robin Cleveland robin.cleveland@eng.ox.ac.uk University of Oxford 

Louise Coletta P.L.Coletta@leeds.ac.uk University of Leeds 

Michael Conneely m.j.conneely@dundee.ac.uk University of Dundee 

Kevin Critchley K.Critchley@leeds.ac.uk University of Leeds 

Paul Dayton padayton@bme.unc.edu University of North Carolina 

Nico de Jong n.dejong@erasmusmc.nl Erasmus MC 

Pratik Desai pratikdr@gmail.com University of Sheffield 

Mohan Edirisinghe m.edirisinghe@ucl.ac.uk University College London 

Stephen Evans S.D.Evans@leeds.ac.uk University of Leeds 

Tony Evans J.A.Evans@leeds.ac.uk University of Leeds 

Joe Fiabane f.j.fiabane@rgu.ac.uk Robert Gordon University, 

Aberdeen 

Steven Freear S.Freear@leeds.ac.uk University of Leeds 

Chris Fury chris.fury@npl.co.uk National Physical Laboratory 

Sevan Harput eensha@leeds.ac.uk University of Leeds 

George Heath py06gh@leeds.ac.uk University of Leeds 

John Hossack hossack@virginia.edu University of Virginia 

Nicola Ingram n.ingram@leeds.ac.uk University of Leeds 

Fairuzeta Ja'afar fairuzeta.jaafar06@imperial.ac.uk Imperial College London 

Benjamin Johnson b.r.g.johnson@leeds.ac.uk University of Leeds 

Pam Jones P.Jones@leeds.ac.uk University of Leeds 

Jithin Jose jjose@visualsonics.com Visualsonics 

Tom Kokhuis t.kokhuis@erasmusmc.nl Erasmus MC 

Klazina Kooiman k.kooiman@erasmusmc.nl Erasmus MC


16 th & 17 th July 2012 

Vasileios Koutsos vasileios.koutsos@ed.ac.uk University of Edinburgh 

Dmitriy Kuvshinov d.kuvshinov@sheffield.ac.uk University of Sheffield 

Gael Leaute elgyvl@leeds.ac.uk University of Leeds 

Marjorie Longo mllongo@ucdavis.edu UC Davis 

Jonathan Loughran j.loughran09@imperial.ac.uk Imperial College London 

Alex Markham A.F.Markham@leeds.ac.uk University of Leeds 

Gemma Marston G.Marston@leeds.ac.uk University of Leeds 

Jono McKendry jonomckendry@gmail.com University of Leeds 

James McLaughlan J.R.McLaughlan@leeds.ac.uk University of Leeds 

Carmel Moran Carmel.Moran@ed.ac.uk University of Edinburgh 

Chris Newman c.newman@sheffield.ac.uk University of Sheffield 

Olaoluwa Olagunju eloo@leeds.ac.uk University of Leeds 

Sally Peyman S.Peyman@leeds.ac.uk University of Leeds 

Linsey Phillips linsey@live.unc.edu University of North Carolina 

Ben Raiton elbdr@leeds.ac.uk University of Leeds 

Tim Ryan Tim.Ryan@epigem.co.uk Epigem Limited 

Sara Shakil Qureshi sara.shakil@seecs.edu.pk University of Sciences and 

Technology, Pakistan 

Jane Smith Janes.Bates@leedsth.nhs.uk St James's University Hospital, 

Leeds 

Peter Smith efy3prs@leeds.ac.uk University of Leeds 

Mengxing Tang mengxing.tang@imperial.ac.uk Imperial College London 

Neil Thomson N.H.Thomson@leeds.ac.uk University of Leeds 

Michel Versluis m.versluis@utwente.nl University of Twente 

Charles Winckler University of Leeds

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