Microbubble Symposium: Fabrication, Characterisation and ...

Microbubble Symposium: 

Fabrication, Characterisation and Translational 

Applications 

4 th & 5 th July 2011 

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

2 Microbubble Symposium: Fabrication, Characterisation and Translational Applications 

4 th & 5 th July 2011

Microbubble Symposium: Fabrication, Characterisation and Translational Applications 3 

4 th & 5 th July 2011 

Contents 

Program 4 

Abstracts: 

Page 

Oral presentation – Session 1 

Microbubble Fabrication and Characterisation 7 


Microbubble Ultrasound Characterisation 13 


Microbubble Translational Applications 19 

Poster presentations 24 

Delegate list 46


4 th & 5 th July 2011 

Monday 4 th July 2011 

Program 

12:00 – 14.00 Registration and buffet lunch 

14:00 – 14:15 Stephen Evans Welcome 

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

14:15 – 14:50 Marjorie Longo Physical chemistry of lipid/lipopolymer shelled 

models of medical microbubbles 

14:50 – 15:10 Klazina Kooiman A novel method to determine the lipid viscosity 

and distribution of coated microbubbles 

15:10 – 15:40 Tea / Coffee 

15:40 – 16:15 Eleanor Stride Magnetic microbubbles for localised imaging 

and drug delivery: development, 

characterisation and preliminary application in 

vivo 

16:15 – 16:35 Jonathan McKendry Mechanical properties of surface modified 

microbubbles by atomic force microscopy 

16:35 – 17:10 Ine Lentacker Update on microbubble and ultrasound assisted 

drug delivery within the Sonodrugs project 

18:00 – 19:30 Poster Session and Drinks 

19:30 - onwards Symposium Dinner


4 th & 5 th July 2011 

Tuesday 5 th July 2011 

Session 2: Microbubble Ultrasound Characterisation – Chair: Steven Freear 

09:00 – 09:35 Vassilis Sboros Single microbubble acoustics 

09:35 – 09:55 Verya Daeichin Stimulating the subharmonic response of 

ultrasound contrast agents using selfdemodulation 

09:55 – 10:30 Robert Eckersley Quantitative contrast-enhanced ultrasound 

imaging: pitfalls, challenges, and advances 

10:30 – 11:00 Tea / Coffee 

11:00 – 11:20 Serge Mensah Release and characterization of a single bubble 

11:20 – 11:55 Paul Campbell Observations of microbubble cavitation under 

optical trap control 

12:00 – 13:30 Lunch 

Session 3: Microbubble Translational Applications – Chair: Louise Coletta 

13:30 – 14:10 Fabian Kiessling Microbubbles for molecular imaging and 

theranostics 

14:10 – 14:30 Gemma Marston Imaging the development of functional tumour 

vasculature in vivo in preclinical models 

14:30 – 14:50 Philippe Trochet VevoCQ software allows advanced 

quantification of perfusion kinetics parameters 

with consistent, reliable and actionable data 

14:50 – 15:20 Tea / Coffee 

15:20 – 15:55 Sarah Fawcett Our early experience of pre clinical in vivo use of 

MicroMarker 

15:55 – 16:30 Jane Smith (Bates) Bubbles in the clinical setting 

16:30 – 16:45 Sir Alex Markham Closing remarks


4 th & 5 th July 2011


4 th & 5 th July 2011 

Session 1: Microbubble Fabrication and Characterisation 

Oral Presentation 

Marjorie Longo 

University of California 

Title: Physical Chemistry of lipid/lipopolymer shelled models of medical microbubbles 

Abstract: In this talk, I will reflect upon my group’s investigation of the richness of physical 

chemical behavior in microbubble shells resembling the lipid shells of certain medical 

microbubbles. These findings will then be related to our most recent work attempting to 

understand and engineer the performance of microbubbles. We found that a decrease in 

the permeability of the shell to air took place with increasing saturated acyl chain length and 

decreased disordered domain boundary density, as could be described by a modified energy 

barrier theory that incorporated a simple accessible area model. The morphology of 

microbubble shell collapse structures and shed particles depended upon both reduced 

temperature and area compression rate, a consequence of time-temperature superposition. 

Shell monolayer phase behavior and phase diagrams were strongly dependent upon 

saturated acyl chain length of the major component and number of chains in the minor 

emulsifier component (mono-acyl vs. di-acyl) and included discovery of a stabilised 

condensed stoichiometric complex (diC14PC3DSPE-PEG20001). Similarly, the bi-acyl 

emulsifier (lipopolymer) conferred on the shells 10X higher resistance to gas permeability, a 

consequence of the reduced disordered boundary density in the presence of this condensed 

component. These results should shed light upon formulation and performance of lipidshelled 

medical microbubbles. To demonstrate, I will end the talk briefly with some of the 

latest observations with respect to maintaining monodispersity, attachment/detachment of 

drug delivery vesicles, and microbubbles formed at the “optimum” stoichiometric complex 

composition.


4 th & 5 th July 2011 



Klazina Kooiman 

Thoraxcenter, Erasmus MC 

Title: A novel method to determine the lipid viscosity and distribution of coated 

microbubbles 

Abstract: Ultrasound contrast agents consist of gas-filled coated microbubbles (MB) with diameters 

between 1 and 10 µm. Within an ultrasound field, large differences in responses of similar sized MBs 

have been reported1,2. Heterogeneous coating properties have been suggested to be the underlying 

cause. Until now, properties of this coating, like viscosity have been studied dynamically using a setup 

of vibrating MBs in an ultrasound field. This study focuses on determining the viscosity of the 

coating for lipid-coated MBs in a static set-up. For that we used MBs with a C4F10 gas core and a 

coating mixture of DSPC (59.4 mol %), PEG-40 stearate (35.7 mol%), DSPE-PEG(2000) (4.1 mol%) and 

DSPE-PEG(2000)-biotin (0.8 mol%). MBs were made by sonication3, and fluorescent streptavidin was 

conjugated4. The viscosity of the lipid coating was determined by measuring the mobility of the 

fluorescent lipid using Fluorescence Recovery After Photobleaching (FRAP). Recordings were 

acquired before and after photobleaching a part of the MB coating. The speed of recovery formed 

the input of a Monte Carlo simulation resulting in the diffusion coefficient of the fluorescent lipid. 

The surface shear viscosity was analytically derived from the diffusion coefficient. We found a surface 

shear viscosity of 8×10-6 kg/s that was independent of the MB size, in line with values reported for 

the dilatation viscosity in MB dynamics studies5. In addition, we found heterogeneous lipid 

distributions in the coating which varied between MBs (see Figure) which suggests partly 

immiscibility of the lipids. In conclusion, this study shows that the static surface shear viscosity of the 

coating can be determined in an independent way which can now be used in MB dynamics models. 

References: 

1 Emmer, M., PhD thesis: The onset of bubble vibration, 2009; 2 Faez, T. et al., IEEE Ultras. Symp. Proc. 2010; 

3 Klibanov, A.L. et al., Invest. Radiol., 2004. 39: 187-95; 4 Lindner, J.R. et al., Circulation, 2001. 104: 2107-12; 5 van 

der Meer, S.M. et al., J. Acoust. Soc. Am., 2007. 121: 648-56. 

Additional authors: Klazina Kooiman *a , Marcia Emmer a , Tom J.A. Kokhuis a,b , Johan G. Bosch a , H. 

Martijn de Gruiter c , Martin E. van Royen c , Wiggert A. van Cappellen d , Adriaan B. Houtsmuller c , 

Antonius F.W. van der Steen a,b , Nico de Jong a,b,e 

a Biomedical Engineering, c Pathology, d Reproduction and Development, Erasmus MC, the Netherlands; b ICIN, 

the Netherlands; e Physics of Fluids Group, Univ. of Twente, the Netherlands 

Figure. Lipid distribution of coating of MBs recorded by 4Pi microscopy. Two typical views (1 and 2) out of a 

3D acquisition for a 3.0 µm (a) and 7.9 µm (b) MB. DSPE-PEG(2000)-biotin was labelled with fluorescent 

streptavidin whereas the other coating components DSPC and PEG-40 stearate were not fluorescently labelled.


4 th & 5 th July 2011 



Eleanor Stride 

University College London 

Title: Magnetic microbubbles for localised imaging and drug delivery: development, 

characterisation and preliminary application in vivo 

Abstract: Coated microbubbles have now been in clinical use as contrast agents for 

ultrasound imaging for several decades. More recently, their use in therapeutic applications, 

in particular drug delivery and gene therapy, has also become a highly active area of 

research. There remain, however, some significant challenges which must be overcome in 

order to fully realise the potential of microbubbles in these applications. In particular, the 

difficulty in controlling the concentration of microbubbles at a given site and in ensuring 

sufficient proximity between bubbles and target cells, has frequently led to disappointing 

results from in vivo studies. Recent work has indicated that incorporating magnetic 

nanoparticles into the microbubble coating may provide an effective strategy for 

overcoming these challenges, by both enabling the bubbles to be localised using an 

externally applied magnetic field and enhancing the efficiency of cell uptake [1][2]. 

Significant improvements in gene transfection efficiency have been demonstrated in vitro. 

Further investigation to fully understand the mechanisms of enhancement and hence 

optimise the delivery protocols is however required and the present study is concerned with 

the detailed characterisation of the magnetic microbubbles and their application for gene 

transfection in vivo. The results from optical, acoustic and magnetic characterisation will be 

presented, including the successful manipulation of suspensions of magnetic bubbles subject 

to physiological flow conditions. Videos from high speed imaging used to investigate the 

dynamic behaviour of single microbubbles and the influence of the magnetic field on their 

response to an acoustic field will also be presented and compared with theoretical modelling 

conducted to support and interpret the experimental findings. Finally results demonstrating 

in vivo transfection in a mouse model will be presented confirming successful localisation of 

the transfection site. 

References: 

[1] Stride, E., Porter, C., Prieto, A. G., Pankhurst, Q. (2009) Ultrasound in Medicine and Biology, 35, 861-868; [2] 

Vlaskou, D., Mykhaylyk, O., Pradhan, P., Bergemann, C., Klibanov, A. L., Hensel, K., Schmitz, G., and, C. (2010) 

Human Gene Therapy, 21, 1429-1430


4 th & 5 th July 2011 



Jonathan McKendry 

University of Leeds 

Title: Mechanical properties of surface modified microbubbles by atomic force microscopy 

Abstract: Atomic force microscopy (AFM) has been used to investigate the mechanical 

properties of phospholipid coated microbubbles and to quantify their stiffness. The 

mechanical properties were investigated using tipless AFM cantilevers to compress 

microbubbles attached to a gold surface in aqueous conditions. The phospholipid 

microbubbles were produced by microfluidic flow focusing and were found to have stiffness 

of 25 mNm -1 . The attachment of a streptavidin coating increased the microbubble stiffness 

by a factor of 30 to around 750 mNm -1 . Further, estimation of the frequency response based 

on values of stiffness obtained by force spectroscopy seem reasonable in comparison with 

those of an uncoated bubble and a PEG coated Bracco SonoVue BR14, suggesting that it may 

provide useful information in the development of novel microbubble coatings. It is hoped 

that the combination of AFM for measuring properties coupled with protein attachment for 

frequency response and targeting will lead to new design rules for the formulation of 

therapeutic microbubble contrast agents. 

Figure 1: Schematic showing a microbubble attached to a gold substrate via a biotin-streptavidin bridge and 

being indented by a tipless AFM cantilever. 

Additional authors: Mr J. E. McKendry 1 , Dr C. Grant 1 Dr P. L. Coletta 2 , Dr J. A. Evans 3 , Prof S. 

D. Evans 1 

1. School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT; 2. Leeds Institute of Molecular 

Medicine, Wellcome Trust Brenner Building, St James’s Hospital, Leeds, LS9 7TF; 3. Leeds Institute of Genetics, 

Health and Theraputics, Worsley Building, University of Leeds, Leeds, LS2 9JT


4 th & 5 th July 2011 



Ine Lentacker 

Ghent University 

Title: Update on microbubble and ultrasound assisted drug delivery within the Sonodrugs 

project. 

Abstract: The Sonodrugs project is an EU FP7 collaborative research project (NMP-4-LA- 

2008-213706) consisting of 14 partners. The objective of this project is to develop new drug 

carriers which can be activated for local drug release using focused ultrasound. One part of 

the project focuses on the design of pressure sensitive drug carriers (microbubbles) which 

can be imaged and locally activated with ultrasound. Another part focuses on thermosensitive 

drug carriers which can be activated by local heating using focused ultrasound 

under MRI image guidance. 

In this presentation we would like to give an overview of the results on microbubble and 

ultrasound based drug delivery within the project. Firstly, we will present some data on the 

co-administration of drugs with polymer and lipid microbubbles using doxorubicin (DOX) and 

Evans Blue as model drugs (1). These results clearly show enhanced drug delivery and 

ultrasound induced extravasation in vitro and in vivo. In a second part, we discuss our work 

on the development of a pressure sensitive liposome loaded microbubble. We succeeded in 

coupling DOX containing liposomes (Doxil®-like) onto lipid microbubbles. In vitro data 

showed that this system is very efficient to enable DOX release and enhance DOX delivery 

upon ultrasound exposure. This was confirmed with a cell viability assay which showed that 

ultrasound exposure of DOX-liposomes attached to microbubbles resulted in a 3-fold higher 

toxicity compared to the DOX-liposomes alone (2). We further optimized this concept to 

obtain an easy to use, sterile and non-immunogenic formulation and proved that even very 

low DOX-liposome doses are sufficient to promote tumor cell killing after microbubble 

loading and ultrasound exposure (3). Furthermore this self-assembled liposome loaded 

microbubble system was characterized with the Brandaris microscope, showing expansion 

only behaviour of liposome loaded microbubbles even at low acoustic pressures. In vivo 

experiments with this new drug carrier are currently ongoing. 

References: 

(1) Böhmer M. et al. Focused ultrasound and microbubbles for enhanced extravasation. Journal of Controlled 

Release 2010, 148 (1), 18-24; (2) Lentacker I., et al. Design and evaluation of doxorubicin containing 

microbubbles for ultrasound triggered doxorubicin delivery: cytotoxicity and mechanisms involved. Molecular 

Therapy 2010, 18(1), 101-108; (3) Geers B., et al. Self-assembled liposome loaded microbubbles: The missing 

link for safe and efficient ultrasound triggered drug delivery. Journal of Controlled Release 2011, 152(2), 249- 

256. 

Additional authors: I. Lentacker 1 , B. Geers 1 , S. C. De Smedt 1 , M. Mühlmeister 2 , K. Tiemann 2 , Y. 

Luan 3 , N. De Jong 3 , J.-M. Escoffre, A. Bouakaz, M. Böhmer 5 , S. Vulto 5 , C. F. Sio 5 

1 Ghent Research Group on Nanomedicines, Ghent University, Belgium; 2 Westfälische Wilhelms-Universität 

Münster, Germany; 3 Erasmus MC Rotterdam, The Netherlands.; 4 Université Francois Rabelais, Tours, France. 

5 Philips Research Europe, Eindhoven, The Netherlands.


4 th & 5 th July 2011


4 th & 5 th July 2011 

Session 2: Microbubble Ultrasound Characterisation 


Title: Single microbubble acoustics 

Vassilis Sboros 

University of Edinburgh 

Abstract: Ultrasound microbubble (MB)-enhanced imaging is currently applied in the clinic 

for heart and liver diagnosis. The potential use of quantifying microvascular flow has been 

researched for over 20 years. More recent research has explored novel applications of MB 

technologies such as molecular imaging, enhancement of cell porosity, thrombolysis and 

drug/gene delivery. The slow progress of the field may be attributed to the lack of high 

quality experimental data that enable a thorough understanding of MB behaviour under 

realistic ultrasound fields and in realistic in vivo experimental settings, which in turn will 

facilitate translation of research into novel in vivo tools. 

The necessity for investigating the acoustics of single MBs stems from the lack of a single or 

a predictable distribution of their acoustic responses. In other words investigations of MB 

clouds are limited in providing information on the individual scatter components, thus 

making difficult the comparison of experimental and theoretical data, but also an 

assessment of the performance of signal processing algorithms. Single MB acoustics 

measurements have provided high quality data that may advance MB theory and signal 

processing research. 

With the help of accurate calibration of MB scatter it is possible to observe and study 

physical phenomena such as resonance, the onset of transient cavitation, MB cracking, the 

different contributions of the shell, gas and environment including narrow tubing, and the 

various decay mechanisms. 

In addition pulse sequences available in commercial scanners can be assessed accurately. It 

is possible to capture large sample sizes of signal distributions and enable thorough signal 

processing analysis without the prerequisite of a model MB behaviour. It has been found 

that current pulse sequences, such as amplitude modulation, have a suboptimal operation as 

they exploit a small proportion of MBs, while they successfully cancel linear tissue echoes at 

low mechanical index imaging. 

In conclusion single MB acoustic measurements offer high quality data for the development 

of signal processing, improve the theoretical understanding of MB behaviour under well 

controlled experimental conditions, and can efficiently characterise different MB types, 

which is useful for the development of MB technologies.


4 th & 5 th July 2011 



Verya Daeichin 

Thoraxcenter, Erasmus MC 

Title: Stimulating the subharmonic response of ultrasound contrast agents using selfdemodulation 

Abstract: Subharmonic (SH) emission from ultrasound contrast agent (UCA) is of interest since this is 

produced only by the UCA and is free of artifacts produced in harmonic imaging modes. In this work 

we study the use of self-demodulation (S-D) signal as a means of microbubble excitation at the SH 

frequency in order to enhance the SH emission of UCA. The S-D wave is a low-frequency signal 

produced by nonlinear propagation of an ultrasound wave in the medium and it can be 

approximated by the second time derivative of the squared envelope of the transmit pulse. A diluted 

population (1:10000) of Definity UCA (Bristol Myers Squibb, Boston, MA, USA) was insonified by a 10 

MHz single element transducer focused at 76mm. The scattered signals were collected by another 10 

MHz transducer. The transmitted 10 MHz wave with a low acoustic pressure of 50 kPa (peak 

negative) had a burst length of 6 and 20 cycles which was multiplied with the envelope function: E( t 

) = exp[- ( 2t/T)^2M] where T is the nominal duration of the pulse and the integer M determines the 

rise and decay time of the envelope (see figure below). The center frequency of the S-D signal is 

changing from low frequencies (around 1 MHz) towards the transmitted frequency (10 MHz) by 

modifying the envelope function from Gaussian (M=1) to rectangular (M=15). The amplitude of S-D 

signal is at its maximum 40 dB below the fundamental transmit amplitude. In order to look at the 

true enhancement of SH emission in frequency domain and knowing that the S-D wave is located at 

the beginning and the end of the received signal, the analysis was performed in a time window 

discarding 3 periods at 10 MHz from the beginning and the end of the 20 cycle burst (see figure 

below). For both 6 and 20 transmitted cycles, the SH response is increased up to 20 dB because of 

the S-D stimulation when M is equal to 15 (rectangular envelope). A suitable design of the envelope 

of the transmit excitation to generate a S-D signal at the SH frequency can enhance the SH emission 

of UCA. This study suggests the SH imaging is feasible with low cycle-number transmit burst and low 

acoustic pressure (~50 KPa). 

Normalized Scattered Pressure (dB) 

0 

-20 

-40 

-60 

-80 

-100 

-120 

Spectrum of the signal scattered by the UCA 

-0.5 

M = 1 

0 0.5 1 1.5 2 2.5 3 

x 10 5 

5 10 15 20 25 

Frequency (MHz) 

Influence of the S-D signal on the spectrum of the signal scattered by UCA insonified by a 20cycle 

burst of 10 MHz. 

This research was supported by the Center for Translational Molecular Medicine and the Netherlands Heart Foundation (PARISK). 

1 

0.5 

0 

-1 

1 

0.5 

0 

M = 3 

-0.5 

Excitation Burst 

0 0.5 1 1.5 2 2.5 3 

x 10 5 

-1 

1 

0.8 

0.6 

0.4 

M = 0.215 

0 

-0.2 

-0.4 

-0.6 

-0.8 

-1


4 th & 5 th July 2011 



Robert Eckersley 

Imperial College 

Title: Quantitative contrast-enhanced ultrasound imaging: pitfalls, challenges, and advances. 

Abstract: Ultrasound imaging of microbubble contrast agents provides the potential to 

obtain quantitative information relating to blood supply and tissue function. This technique 

is showing great promise for diagnosis and monitoring clinical conditions such as 

cardiovascular diseases and cancer, with considerable potential benefits in terms of patient 

care. 

A key challenge of this technique is the existence of significant variations in the imaging 

results, and the lack of understanding regarding their origin. 

In this presentation the potential sources of variability in the quantification of tissue 

perfusion based on microbubble contrast-enhanced ultrasound images will be presented 

and discussed. These are divided into the following three categories: (i) factors relating to 

the set up of the ultrasound scanner, including transmission power, focal depth, dynamic 

range, signal gain and ultrasound frequency, (ii) factors relating to the patient, which include 

body physical differences, physiological interaction with the microbubbles, propagation and 

attenuation through tissue, and tissue motion, and (iii) factors relating to the microbubbles, 

including the type of bubbles and their stability, preparation, injection technique and 

dosage. 

It has been shown that the factors in each category can significantly affect the imaging 

results and contribute to the variations observed. How these factors influence quantitative 

imaging is explained and possible methods for reducing such variations are discussed. 

Additional authors: R. J. Eckersley 1 , E. Stride 4 , H. Mulvana 1 , T. Gauthier 3 , A. K. P. Lim 1 , D. O. 

Cosgrove 1 , and M.-X. Tang 2 

1 Imaging Sciences Department, Faculty of Medicine, Imperial College London. 2 Department of Bioengineering, 

Imperial College London. 3 Department Experimental Medicine and Toxicology, Imperial College London. 4 

Department Mechanical Engineering, University College London.


4 th & 5 th July 2011 



Title: Release and characterization of a single bubble 

Serge Mensah 

CNRS 

Abstract: During hyperbaric decompression (diving or hyperbaric medicine), both the absolute 

ambient and the absolute inspired pressures are reducing; (5-100 µm) bubbles may be generated 

from pre-existing gas nuclei (0.1-5 µm). An accurate monitoring of the size and of the density of the 

bubble (including the nuclei) population will provide a valuable means to understand the nucleation 

and growth processes in various supersaturated tissues. In this aim, an ultrasonic characterization 

method based on a dual frequency technique [1] applied on a free single bubble is tested. 

In order to design and evaluate the accuracy of a free single bubble sizing technique operating in 

water, bubbles of different diameters tethered on a wire have been set free by using high amplitude 

pressure waves. Then, two ultrasound focused waves impinge simultaneously on each microbubble: 

a low frequency chirp (20 kHz


4 th & 5 th July 2011 



Title: Observations of microbubble cavitation under optical trap control 

Paul Campbell 

University of Dundee 

Abstract: Microbubbles are held up as a potential next generation 'theranostic' agent - 

exhibiting clear therapeutic characteristics in terms of their ability to permeabilize tissue and 

individual cell membranes for the purposes of either drug delivery or to induce distinct 

bioeffects, but also retaining a critical diagnostic capability whilst in their intact state. We 

have been studying microbubbles at Dundee for several years now, initially using optical trap 

control to isolate single microbubbles for precision observation at distinct proximities from 

individual cells. More recently, we have attempted to seed spatially controlled free bubble 

cavitation events, for the purposes of achieving enhanced transfection in vitro, via the laser 

ignition of optically trapped nanoparticles. 

This talk will thus serve as an introduction to the optical trap approach to cavitation in 

general, so that others might easily replicate our procedures, and will also provide an 

overview of the main results that we have achieved for both ultrasonically activated shelled 

microbubbles, as well as free microbubbles generated via laser ignition of optically trapped 

nanoparticles.


4 th & 5 th July 2011


4 th & 5 th July 2011 

Session 3: Microbubble Translational Applications 


Title: Microbubbles for molecular imaging and theranostics 

Fabian Kiessling 

University of Aachen 

Abstract: Ultrasound is one of the workhorses in clinical cancer diagnosis. In particular, it is 

routinely used to characterize lesions in liver, urogenital tract, head and neck and soft 

tissues. Microbubbles were introduced as intravascular contrast agents and can be detected 

with superb sensitivity and specificity using contrast specific imaging modes. By conjugating 

biomolecules to the microbubble surface molecular ultrasound imaging becomes feasible. 

In this talk our experiences in fabricating and using microbubbles for targeted imaging and 

for theranostics are reported. Examples will be given for the use of soft and PBCA-based 

hard-shell MBs for tumour characterisation and therapy response monitoring. For example, 

it will be shown that the assessment of the VEGFR2-expression on the tumour 

vascularisation is better suited for the discrimination of low and high aggressive breast 

cancers compared to functional information about the relative tumour blood volume. On the 

other hand, it will also be shown that the normalisation of the molecular ultrasound data to 

the relative blood volume often is necessary to distinguish between change in vessel density 

and change in molecular marker expression on the vasculature. However, the use of 

microbubbles is not restricted to ultrasound. By incorporating dyes and superparamagentic 

iron oxide nanoparticles into the microbubble shell multimodal diagnostics can be achieved. 

In this context, we will show that these microbubbles can excellently be detected by MRI 

and that their disintegration by ultrasound leads to a changed MR contrast. It will further be 

shown that contrast-enhanced ultrasound mediated vascular permeation can favourably be 

monitored with these multimodal probes. In summary, microbubbles are versatile diagnostic 

and theranostic agents, which can be used not only for ultrasound but also for optical 

imaging and MRI.


4 th & 5 th July 2011 



Gemma Marston 


Title: Imaging the development of functional tumour vasculature in vivo in preclinical models 

Abstract: 

Background 

Angiogenesis is essential for tumours to grow beyond a few millimetres in size and to metastasise, 

making it an attractive target for both cancer imaging and therapeutic intervention. 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 to characterise the 

development of functional tumour vasculature in vivo in a murine xenograft model of colorectal 

cancer. 

Method 

10 female CD1 Nu/Nu mice implanted with SW480 human colorectal cancer cell xenografts were 

imaged with CE HFUS 7 times between day 7 and day 28 of tumour growth using the VisualSonics 

Vevo 770 system. At each time point a single bolus of microbubbles was injected using a syringe 

driver into the tail vein during 2D CE HFUS imaging, with the wash-in data loops captured prior to 3D 

CE HFUS imaging. 

Results 

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

vascular data. Tumour vascular space and tumour volume were 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 both faster rates of blood flow and significantly 

higher maximum levels of vascular perfusion than larger tumours. 

Conclusion 

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 therapies 

and response to treatment in pre-clinical trials.


4 th & 5 th July 2011 



Philippe Trochet 

VisualSonics 

Title: VevoCQ Software allows advanced quantification of perfusion kinetics parameters with 

consistent, reliable and actionable data 

Abstract: VevoCQ is a powerful addition to nonlinear contrast imaging functionality on the 

Vevo® 2100 imaging system. This software allows for the study of contrast uptake kinetics as 

well as late phase targeted enhancement. It provides advanced curve fitting algorithms for 

quantitative assessment of perfusion parameters as well as color-coded parametric images 

useful for qualitative assessment of the spatial distribution of the same parameters. The 

software is a post-processing tool which can be used on numerous tissues, organs and tumor 

models, including subcutaneous tumors, abdominal organs and hind limb muscles.


4 th & 5 th July 2011 



Sarah Fawcett 

University of Cambridge 

Title: Our early experience of pre clinical in vivo use of MicroMarker – a) sonoporation for in 

vivo gene delivery and b) contrast enhanced imaging in tumours after anti-vascular therapy 

Abstract: We have attempted to use MicroMarker mediated sonoporation for in vivo 

delivery of DNA. Using this approach I will present our pilot data in which we demonstrate 

sustained expression of a gene reporter, several weeks after DNA injection into the skin. 

In addition we have started to investigate the potential for contrast enhanced ultrasound 

imaging for monitoring tumour response to anti-vascular agents and comparing these data 

with those obtained from DCE-MRI imaging.


4 th & 5 th July 2011 



Title: Bubbles in the clinical setting 

Jane Smith (Bates) 

St James’s University Hospital 

Abstract: US contrast agents are now an important part of the diagnostic process, and a 

recognised problem-solving tool. 

The main (non-cardiac) use hinges around lesion characterisation in the liver, with patterns 

of contrast take-up allowing the operator to establish a definitive diagnosis. Detection of 

malignant lesions in the liver is also a useful application, particularly in patients at high risk 

of liver metastases. Other uses include trauma, monitoring of treatments (such as ablation) 

and guiding targeted biopsies in the liver. 

The advantages of bubbles in the clinical field are established; it is a safe, cost effective and a 

reliable addition to our available diagnostic tools. It has high patient acceptability and, 

importantly, allows the operator to make a diagnosis which is immediately available - 

shortening the diagnostic pathway and reducing or eliminating patient anxiety. 

This talk will outline some of the common clinical applications and current capabilities of 

commercially available bubbles.


4 th & 5 th July 2011 

Number Name Title 

1 Mostafa Abdelrahman 

2 Radwa Abou-Saleh 

3 Muhammad Arif 

4 Jonathan Blockley 

5 Matthew Booth 

6 Christian Buchcic 

7 Joanne Burke 

Poster Presentations 

Optimizing contrast-enhanced ultrasound technique for 

mice tumour imaging: choosing the right transducer 

and bubble concentration 

Probing the effect of poly(ethylene glycol) on the 

mechanical behavior of streptavidin and quantum dot 

coated microbubbles 

Chirp coded excitation with fractional Fourier transform 

for ultrasound harmonic imaging 

Ultrasound measurements of trapped microbubbles on 

a standard microscope stage 

The optical properties and stability of cadmium free 

quantum dots 

Microbubbles as functional ingredients in complex food 

systems 

Interfacial study of class II hydrophobin and β-casein 

mixtures: relationship to bubble stability 

8 Caroline Harfield Opto-acoustic trapping of microbubbles 

9 Sevan Harput 

10 Sevan Harput 

11 George Heath Controlling actin filaments 

12 Nicola Ingram 

13 Gaël Léauté 

Periodic clustering of microbubbles by secondary 

radiation force 

Effect of nonlinear frequency modulated signals on 

subharmonic emission from microbubbles 

Pre-clinical evaluation of VEGFR2 as an imaging 

biomarker and targeting molecule for therapeutic 

delivery. 

Effect of excitation pulses on the sonoporation 

efficiency of mono- and polydisperse ultrasound 

contrast agents


4 th & 5 th July 2011 

14 Jonathan Loughran 

15 Paul Mackin 

16 Richard O'Rorke 

17 Sally Peyman 

18 Benjamin Raiton 

19 Charles Sennoga 

20 Peter Smith 

Selective imaging of adherent microbubbles with 

correction of tissue motion 

Non-Invasive ultrasound imaging in a mouse model of 

pancreatic ductal adenocarcinoma 

Trapping microbubbles in a moveable acoustic array 

using surface acoustic waves 

3D expanding geometry for improved on-chip 

microbubble production rates 

Counter flow microbubble channelling using acoustic 

radiation force funnel 

Comparision of methods for sizing and counting of 

microbubbles 

Pre-distorted chirp excitations for polydisperse 

microbubble populations


4 th & 5 th July 2011 

Poster Number: 1 

Mostafa Abdelrahman 


Title: Optimizing contrast-enhanced ultrasound technique for mice tumour imaging: 

choosing the right transducer and bubble concentration 

Abstract: High-frequency contrast-enhanced ultrasound (HF-CEUS) has been shown to be a 

valuable tool in the assessment of tumour angiogenesis in mice. However, this technique is 

still to be optimized and calibrated. Forty MHz is the most commonly used frequency in mice 

CEUS imaging due to the high spatial resolution it provides. However, 25 MHz frequency 

provides more penetration power and is closer to the resonance frequency of bubbles of the 

sizes used in mice imaging (~3MHz). In this study the response of 40 MHz and 25 MHz 

transducers to contrast was investigated. 

Both transducers were used to image contrast added to blood-mimicking fluid (BMF) at 

different concentrations (5 x 10 5 - 5 x 10 7 bubble/ml). The contrast-BMF mixture was driven 

through a flow phantom using an infusion pump at flow velocities of 1, 2, and 3 mm/s. 

Greyscale intensity was used as a measure for signal enhancement from bubbles. 

The results showed that the 25 MHz was 1.5 times more sensitive to contrast than the 40 

MHz. It was also found that the effect of increasing contrast concentration is less important 

at concentrations more than 1.25 x 10 7 bubbles/ml for both transducers. Finally, it was 

shown that flow velocity has no significant effect on contrast detectability. 

In conclusion, based on the results of this study it is recommended to use ultrasound at 25 

MHz frequency instead of 40 MHz in CEUS studies, and it is possible to use lower bubble 

concentrations without significantly reducing the signal.


4 th & 5 th July 2011 


Radwa Abou-Saleh 


Title: Probing the effect of poly(ethylene glycol) on the mechanical behavior of streptavidin 

and quantum dot coated microbubbles 

Abstract: The use of microbubbles as targeted delivery vehicles has been receiving wide 

spread interest for a range of medical applications. Potentially ultrasound can be used for 

localized delivery that is by rupturing bounded microbubbles in a specific area and introduce 

payloads into cells via sonoporation. The frequency response of microbubbles to ultrasound 

excitation mainly depends on the mechanical properties of the shell[1] which can be 

determined using AFM[2]. Here, we perform force spectroscopy measurements-with tipless 

cantilever on encapsulated microbubbles to characterize the shell stiffness of a range of 

microbubble coatings, specifically, Streptavidin and Quantum dot coated microbubbles. 

Forces ranging from 20-100nN were applied to single bubbles to determine the change in 

stiffness with applied force. In addition, the same coatings were tested in the presence and 

absence of a Poly(ethylene glycol) (PEG) layer with the same protocol. It was observed that 

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

Adding a streptavidin coat doubles the stiffness values of the microbubble, with a dramatic 

increase observed with addition of Quantum dots leading to a 3x increase in shell stiffness. 

Moreover, PEG-ylating the bubble shell led to distinct compression behaviour in the forcedistance 

curve and presents a characteristic non-linear increase of stiffness in the presence 

of streptavidin and Quantum dot coatings. 

References: 

1. Ferrara, K., R. Pollard, and M. Borden, Ultrasound microbubble contrast agents: fundamentals and 

application to gene and drug delivery. Annu Rev Biomed Eng, 2007. 9: p. 415-47. 

2. Sboros, V., et al., Nanomechanical probing of microbubbles using the atomic force microscope. 

Ultrasonics, 2007. 46(4): p. 349-54. 

Additional authors: 

Radwa H. Abou-Saleh, Jono E. McKendry, Sally A. Peyman, Neil H. Thomson and Stephen D. 

Evans 

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

Phopholipid molecule 

PEG chain 

Biotin molecule


4 th & 5 th July 2011 


Muhammad Arif 


Title: Chirp coded excitation with fractional Fourier transform for ultrasound harmonic 

imaging 

Abstract: Medical ultrasound imaging techniques such as tissue harmonic imaging and 

contrast-enhanced harmonic imaging provides better spatial and contrast resolution by 

producing the image with the second harmonic component (SHC) of the nonlinear received 

signal. The extraction of the SHC from the received signal can be done by linear band-pass 

filtering technique; however it works well only for a narrow-bandwidth signal in which the 

SHC is not overlapped with the fundamental component. A multi-pulse detection scheme 

such as pulse inversion (PI) can be used to extract the overlapped SHC; however it suffers 

under motion artifacts and reducing the system frame-rate. In this paper, fractional Fourier 

transform (FrFT) is proposed as a filtering tool, with wide-bandwidth chirp coded excitation, 

for the extraction of the overlapped SHC. 

Simulation and experiments were performed in order to validate the proposed method. A 

Hann windowed linear chirp with duration of 10 us, and bandwidth ranging from 1-3 MHz 

was used as an excitation signal. The nonlinear received signals were processed using the 

FrFT in order to extract the SHC. The extracted SHC is then processed using the harmonic 

matched filter in order to perform second harmonic pulse compression (SHPC) and to 

restore the axial resolution. 

Both simulation and experimental results indicate at least a 13 dB improvement in the range 

sidelobes level of the FrFT filtered compressed SHC when compared with the unfiltered 

compressed SHC as shown in the enclosed figure. 


Muhammad Arif, Sevan Harput, Peter Smith, David Cowell, and Steven Freear 

Ultrasound Group School of Electronic and Electrical Engineering, University of Leeds, Leeds LS2 9JT


4 th & 5 th July 2011 


Jonathan Blockley 

Institution: University of Leeds 

Title: Ultrasound measurements of trapped microbubbles on a standard microscope stage 

Abstract: It can be difficult to be certain what the size and position distributions of 

microbubble populations are when making ultrasound measurements. Depending on the 

method of production, a broad spread of sizes can result in a small portion of the population 

dominating an ultrasound signal, so unpicking the effects of the spread of sizes is a 

challenge. Existing methods to negate these population effects, or at least obtain repeatable 

population distributions include mixing and pumping through tubes to simulate transport in 

the vascular system, both of which involve fluid motion. Alternatively, clear results can be 

obtained by testing single bubbles; this technique requires very high sensitivity and 

extrapolation of data to the population response. Secondary issues in both cases include the 

methods of bubble isolation, which include confinement in tubes, optical trapping and use of 

ultrasound radiation force. Some of these methods introduce complex boundary effects. 

Here, an apparatus has been designed to allow ultrasound measurements to be made on a 

small population of microbubbles that will be simultaneously imaged with a standard upright 

optical microscope. The microbubbles will be contained within a thin slice of fluid and enter 

the observation area from below, making use of their buoyancy force. Ultrasound 

measurements will be made as the microbubbles float through the observation area. The 

optical microscope can be focused at a depth of choice to image the microbubbles as they 

rise, or survey the microbubbles that were insonated after they have risen to the top of the 

chamber. Thus the size distribution and concentration of the microbubbles can be 

ascertained in situ. Bubbles of various sizes could be trapped within the observation area 

and their acoustic responses matched to the observed size distribution of the population. 

This system also has the advantage that the bubbles can be tested at a distance 100 to 1000 

times their radius from any surface, with the absence of boundaries minimising reflections 

and allowing maximum sensitivity.


4 th & 5 th July 2011 


Title: The optical properties and stability of cadmium free quantum dots 

Matthew Booth 


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


4 th & 5 th July 2011 


Title: Microbubbles as functional ingredients in complex food systems 

Christian Buchcic 

Wageningen UR 

Abstract: Besides the utilization of microbubble technology in areas like medical 

engineering, a vast amount of other possible applications can be envisaged. Microbubbles 

have also found attention of the Food Science Community. Our vision is to apply 

microbubbles as functional ingredients in complex food systems in order to modify their 

textural and sensorial properties. The challenge is to find relations between characteristics 

of the shell material, the microbubbles, and their functionality in food systems. 


Christian Buchcic 1, 2 , Tijs Rovers 

2, 3 

1 

Wageningen UR, Physical Chemistry and Colloid Science, P.O. Box 8038, 6700 EK Wageningen, The 

Netherlands 

2 

TI Food and Nutrition, P.O. Box 557, 6700 AN Wageningen, The Netherlands 

3 

Wageningen UR, Physics and Physical Chemistry of Foods, P.O. Box 8129, 6700 EV Wageningen, The 

Netherlands


4 th & 5 th July 2011 


Joanne Burke 


Title: Interfacial study of class II hydrophobin and β-casein mixtures: relationship to bubble 

stability 

Abstract: Class II Hydrophobin (HFBII) is a highly surface active molecule and in the context 

of aeration can be considered to be an air structuring protein conferring exceptional stability 

to foams. Recently, Cox et al [1,2] demonstrated that HFBII can produce liquid foams which 

do not coarsen and are stable for periods of several months, which is far in excess than that 

obtained with any other commonly used proteins. This is of interest to the food industry, 

since producing shelf stable foams in food formulations is very difficult. Although HFBII, 

when used alone, has proven to be very promising in terms of foam stability, a greater 

understanding is required in order to optimise formulations and stability in real food 

systems. Factors which have been explored for this poster include the concentration of HFBII 

(low amounts 10 -4 wt %) as well as the effect of pH on the surface shear viscosity which both 

seem to be very significant. Furthermore, from a practical point of view, mixtures of HFBII 

with other commonly used foaming agents should be tested in order to obtain information 

of more direct relevence to complex food systems since it is unknow as to wether HFBII will 

maintain its functionality in such systems or if synergystic effects will occur. Therefore this 

poster will also describe the surface rheological properties of protein films formed from 

mixtures of HFBII with β-casein. 

References: 

[1] Cox, A.; Cagnol. F.; Russell, A.B.; Izzard, M.J. Langmuir 2007, 23, 7995-8002. 

[2] Cox, A.: Aldred, D.; Russell, A.B. Food Hydrocolloids 2009, 23, 366-376. 


Burke, J 1* , Murray, B 1 , Cox, A 2 and Petkov, J 3 

1 Food Colloids Group, School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK 

2 Unilever R&D, Colworth Science Park, Sharnbrook MK44 1LQ, UK 

3 Unilever R&D, Port Sunlight, Bebingtom CH63 3JW, UK


4 th & 5 th July 2011 


Title: Opto-acoustic trapping of microbubbles 

Caroline Harfield 

University College London 

Abstract: Microbubbbles are used as ultrasound contrast agents for both diagnostic and 

therapeutic applications and are also being investigated for use in gene and drug therapy. 

For the latter, an improved understanding of the response of both individual microbubbles 

and their populations to ultrasound excitation is essential. The challenges associated with 

conventional acoustic experiments on microbubbles have produced irreproducible and often 

contrasting results. In particular, creating a controlled environment in which a single bubble 

can be isolated so that it is unaffected by surrounding bubbles is extremely difficult. 

Therefore a non-contact method for trapping and isolating a single microbubble during 

ultrasound excitation would represent an exceptionally valuable tool. The potential of 

optical tweezers for trapping and manipulating microbubbles have been suggested as a 

suitable confinement method. As a feasibility study, a theoretical model of a single bubble 

exposed to a combination of optical and acoustic fields was developed in order to determine 

the magnitude of the forces acting on upon the bubble. Both the radial and translational 

motion of the bubble were modelled using a Rayleigh Plesset type equation coupled to a 

model for the acoustic radiation force. The results from the simulations indicate the Primary 

acoustic radiation (Bjerknes) force on a typical contrast agent bubble was 3 or more orders 

of magnitude greater that the trapping force which could be provided by the laser, which is a 

trend supported by the existing experimental and theoretical literature. In conclusion, 

classical optical tweezers are not a suitable means of confining acoustically driven bubbles. A 

more sophisticated design, combining the spatial precision of optical trapping with the 

strength of acoustic localisation will be described. 


Caroline Harfield 1,3,* , A Pawlikowska 2,3 , E. Stride 1 , P. H. Jones 2 , G Memoli 3 , 

1- Department of Mechanical Engineering, UCL 

2- Department of Physics and Astronomy, UCL 

3- National Physical Laboratory


4 th & 5 th July 2011 


Title: Periodic clustering of microbubbles by secondary radiation force 

Sevan Harput 


Abstract: Two bubbles excited above or below their resonance frequencies will result in an 

attractive force, while if one is below and the other is above resonance, the net force will be 

repulsive. However, the attractive and repulsive bubble behavior is not always predictable in 

a microbubble cloud. The sign reversal of secondary Bjerknes force acting on microbubble 

clusters is observed due to the multiple scattering effects. Regardless of their sizes, 

microbubbles attract each other and form stable bubble clusters. 

Objective: The mutual interaction between the pulsating bubbles causes the formation of 

the stable bubble clusters due to secondary radiation force. This aggregation pattern of the 

bubbles is also referred as “bubble grapes”. This behaviour of microbubbles can affect the 

results of ultrasound measurements in-vivo and in-vitro. 

Methods: The aggregation behaviour of Sonovue microbubbles are observed at 2 MHz 

between 50 kPa and 500 kPa peak-negative pressures with the flow rates of 1 mL/h to 

10mL/h. A custom design tank is used for the experiments with a 150 micrometer thick 

mylar window for optical observations and a special mount for the transducers. A 

transparent cellulose tube with a diameter of 250 micrometers is placed at the focal point of 

the transducer where the ultrasound contrast agents are flown. The onset of microbubble 

aggregation pattern is captured by Nikon Eclipse Ti-S inverted microscope. 

Results: Under certain conditions, the microbubble aggregation pattern is periodic. The 

attached figure shows the microbubble aggregation pattern for 3 different flow rates and 

pressure levels. 


Sevan Harput, Benjamin Raiton, Dr. James Mclaughlan, Prof. Stephen Evans a and Dr. 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.


4 th & 5 th July 2011 


Sevan Harput 


Title: Effect of nonlinear frequency modulated signals on subharmonic emission from 

microbubbles 

Abstract: Performance of linear frequency modulated (LFM) and nonlinear frequency 

modulated (NLFM) excitation is compared according to their subharmonic emission for nondestructive 

contrast imaging. The subharmonic emission of the microbubbles is measured as 

a function of pressure and bandwidth for a sinusoidal tone-burst, LFM and NLFM signals. 

Objective: The aim of this work is to improve the ultrasound image quality by using coded 

excitation and subharmonic emission from microbubbles. The subharmonic component has 

the potential to improve the contrast-to-tissue ratio (CTR) as it is only generated by the 

microbubble contrast agents. The coded excitation methods increase the signal to noise 

ratio (SNR) and penetration depth by using longer pulse durations. 

Methods: The scattering properties of SonoVue contrast agent were measured in a 

cylindrical chamber containing the 1:1000 diluted SonoVue suspension and mixed with a 

magnetic stirrer during the experiments. An excitation frequency of 5 MHz is chosen to 

excite the microbubbles at twice of their resonance frequency. A 5 MHz transducer mounted 

perpendicular to a 1 mm needle hydrophone was placed 10 mm from the chamber. 

Microbubbles were acoustically excited by a sinusoidal tone-burst, LFM and NLFM signals 

between peak negative pressures of 25-200 kPa. 

Results: It is found that the NLFM signal gives similar or higher subharmonic levels than the 

sinusoidal tone-burst and LFM signal. In average, NLFM excitation with 20% and 40% 

bandwidth achieves 3 dB higher subharmonic levels than LFM excitation. 


Sevan Harput, Dr. Muhammad Arif and Dr. Steven Freear 

Ultrasound Group, University of Leeds, Leeds, UK.


4 th & 5 th July 2011 


Title: Controlling actin filaments 

George Heath 


Abstract: Actin is the most abundant protein found in eukaryotic cells. The 42kDa globular 

protein is a major component of the cytoskeleton as it assembles into filaments producing a 

supporting scaffold beneath cell membranes. As well as providing mechanical support the 

use of actin as a scaffold in biology allows diverse processes such as cell division, cell 

locomotion, muscle contraction and vesicle transportation. This diverse functionality is 

achieved through a huge protein toolbox of actin binding proteins which can be used to: cap, 

cut, crosslink, buddle, branch and move actin filaments. Offering the potential to create 

extremely varied range of structures (either mimicking biology or not) which can be tuned to 

the desired application. We use Atomic Force Microscopy (AFM) in fluid to obtain high 

resolution images of phalloidin stabilized actin filaments electrostaticly bound to Nickel 

treated mica and confirm that controlling relative concentrations of Actin and a 

capping/severing protein Gelsolin tailors filament length.


4 th & 5 th July 2011 


Nicola Ingram 


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

therapeutic delivery. 

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. 

Immunofluorescence using multiple antibodies to VEGFR2 was carried out on endothelial 

cell lines and primary cells. Immunohistochemistry for VEGFR2 expression in vessels was 

carried out on SW480 xenograft tissue. For CE HFUS, microbubbles (MB) were coated with 

VEGFR2 antibody and injected intravenously into SW480-tumour bearing mice. 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 both in 2D 

and in 3D. 

Immunofluorescence studies demonstrated the lack of a suitable murine endothelial cell line 

to use for tumour vascular targeting in pre-clinical studies. 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 

50mm 3 in size. Expression then decreased as the tumours increased in volume. Therefore, 

smaller size xenografts were used for in vivo targeting experiments. VEGFR2-targeted MB 

injection and 3D destruction of bound MBs showed approximately 20% of the tumour 

vasculature in SW480 xenografts had bound microbubbles. 

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

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


Nicola Ingram* 1 , Elizabeth Vallely 1 , Pam Jones 1 , Alex Markham 2 , P Louise Coletta 1 . 

1 Molecular Gastroenterology, 2 Translational Medicine, Leeds Institute of Molecular Medicine, St James’s 

University Hospital, Leeds, U.K.


4 th & 5 th July 2011 


Gaël Léauté 


Title: Effect of excitation pulses on the sonoporation efficiency of mono- and polydisperse 

ultrasound contrast agents 

Abstract: Nyborg 1 theory allows estimations of the shear stress caused by an oscillating 

hemispherical gas bubble on a boundary. Doinikov and Bouakaz 2 further developed this 

model for ultrasound contrast agents (UCA) in the vicinity of a boundary and developed a 

method to estimate the sonoporation efficiency of UCA-cell solution to an ultrasound field. 

This was done with a modified Rayleigh-Plesset equation that incorporated a rigid boundary 

near the oscillating microbubbles. The amount of shear stress exerted by the microbubbles, 

on this boundary, is calculated and by assuming a shear stress threshold of 12 Pa for 

sonoporation to occur, the number of cells affected can be estimated. The sonoporation 

efficiency is then defined as the ratio of the number of sonoporated cells to the total 

number of cells, by assuming cells and microbubbles have the same uniform distribution and 

concentration. It was shown that sonoporation efficiency becomes minimal when the driving 

frequency is equal to the microbubble populations resonance frequency at which 

attenuation of the acoustic field becomes maximal. 

Although the theoretical background of the sonoporation mechanism is still in its infancy, 

our study will use this model as the basis to investigate the effect of ultrasound pulse shape 

on sonoporation efficiency, specifically whether tone bursts or linear swept frequency 

excitation pulses would provide the greatest sonoporation efficiency for mono- and/or 

polydisperse microbubble populations. An experiment could shed light on whether the 

acoustic field attenuation has the strong influence on sonoporation efficiency as described 

by the theoretical results found by Doinikov et al. The ratio of cells which have undergone 

sonoporation could be measured by causing the uptake of a fluorophore into the cell 

through sonoporation induced by different microbubble populations. 

References: 

1 W.L. Nyborg, Acoustic streaming near a boundary, J. Acoust. Soc. Am. 30 (1958) 329-339. 

2 A.A. Doinikov and A. Bouakaz, Theoretical investigation of shear stress generated by a contrast microbubble on 

the cell membrane as a mechanism for sonoporation, J. Acoust. Soc. Am. 128 (2010) 11-19. 


Gaël Y.V. Léauté * , Dr. James Mclaughlan and Dr. Steven Freear 

Ultrasound Group, School of Electronic and Electrical Engineering, University of Leeds, Leeds, UK.


4 th & 5 th July 2011 


Jonathan Loughran 

Imperial College London 

Title: Selective imaging of adherent microbubbles with correction of tissue motion 

Abstract: In order to improve the efficiency of molecular imaging using targeted 

microbubbles, a method is needed to distinguish the free flowing microbubbles from 

adherent microbubbles which have found its predefined targeting site. While simple 

temporal low pass filtering has been used to this end assuming adherent bubbles are 

stationary, this technique would not work in clinical situations when tissue motion is present 

causing movement in the adherent bubbles too. This study aims to address this problem by 

incorporating motion correction into the imaging process. 

In house biotin coated lipid shell microbubbles were inserted into a streptavidin coated 

cellulose tube of 200μm in diameter under steady flow conditions created using a syringe 

pump. The tube located in a water bath was observed optically using a microscopy to verify 

that bubbles were indeed adhering to the surface of the tube, while pulse inversion image 

sequences were simultaneously taken using an ultrasound scanner. Motion was created to 

the tube via a 3D stage holding the tube in place. The image sequence recorded were 

corrected for motion using cross-correlation and the results show that it was possible to 

selectively detect the adherent microbubbles using the low pass filtering techniques after 

the motion correction. 


Jonathan Loughran 1 , Charles Sennoga 12 , Robert Eckersley 2 and Meng-Xing Tang 1 

1 Department of Bioengineering, Imperial College London, South Kensington Campus, London SW7 2AZ, United 

Kingdom; 

2 Imaging Sciences Department, Imperial College London, Hammersmith Campus, London W12 0HS United 

Kingdom;


4 th & 5 th July 2011 


Paul Mackin 

University of Cambridge 

Title: Non-Invasive ultrasound imaging in a mouse model of pancreatic ductal 

adenocarcinoma 

Abstract: Pancreatic ductal adenocarcinoma (PDA) is the fifth most common cause of cancer 

death in the UK causing 7,781 deaths in 2008. Prognosis is extremely poor; survival of 

patients with advanced disease is about 6 months. Despite major efforts in basic and clinical 

research, most therapies still fail to improve survival and novel therapies are urgently 

required for this fatal disease. 

Our lab has produced a genetically engineered mouse model (KPC) carrying Kras and p53 

mutations that recapitulate the clinical and pathological features of human disease including 

site of occurrence, tumours, metastasis, tumour microenvironment, haemorrhagic ascites 

etc. This model is therefore likely to be more predictive of response to novel therapies that 

can then be translated to patients. 

Our ‘Mouse Hospital’ team streamline the production of experimental mice suitable for 

enrolment onto therapeutic studies. Weekly palpation of the mutant mice confirms tumour 

presence. Tumours are measured by non-invasive ultrasound imaging. Mice with a defined 

tumour burden (6-9-mm-diameter) are enrolled onto study. 

We are refining the assessment of pre enrolable tumours by precise palpations and reducing 

the stress of mice being repeatedly ultrasound scanned to confirm and follow the growth 

pattern of the tumours as well as the associated implications of repeated exposure to 

anaesthetic. 

In the past allograft and xenograft models have used countless animals to show therapeutic 

efficacy in the model only, rarely translating efficacy to the clinic. In contrast, we believe that 

the KPC model is a better predictor of response to therapy. Indeed, we have shown that this 

model responds to Gemcitabine, the standard of care for pancreatic cancer, in a similar 

manner as seen in the clinical setting. 

To date, over ten drugs have been tested in the Mouse Hospital. Studies highlighting the 

barrier to drug delivery by the excessive stroma are of particular interest. We have shown 

that the efficacy of chemotherapy can be improved by co-administration of a drug which 

depletes tumour-associated stroma, resulting in an extended lifespan. 

These promising results are currently translated into clinical practice; two clinical trials have 

recently opened to investigate experimental drugs from the Mouse Hospital either in 

advanced or pre-operative PDA patients.


4 th & 5 th July 2011 


Richard O’Rorke 


Title: Trapping microbubbles in a moveable acoustic array using surface acoustic waves 

Abstract: Standing pressure waves at MHz frequencies are used to position and transport 

microbubbles within a microfluidic device. We show that surface acoustic waves (SAWs) – 

nanometer scale vibrations on a piezoelectric crystal – can be excited to form a standing 

wave on the surface, which couples into the liquid channel of an adjacent microfluidic 

device. This results in a standing pressure wave in the liquid, which we use to manipulate 

suspended particles. We discuss the formation of one-dimensional arrays of micron-sized 

latex beads in proving work, in the context of the primary acoustic radiation force. Our 

acoustic alignment technique is applied to a sample of microbubbles and we demonstrate 

microbubble array transport, through small increments in standing SAW frequency. Finally, 

we present two-dimensional particle arrays which could be applied to microbubbles to allow 

individual microbubbles to be spatially controlled for subsequent characterization. 


Richard O’Rorke, C. Wood, C. Walti, A. G. Davies, S. D. Evans, J. E. Cunningham.


4 th & 5 th July 2011 


Title: 3D expanding geometry for improved on-chip microbubble production rates 

Sally Peyman 


Abstract: Micron sized, lipid stabilized bubbles of gas have been utilized in the medical world 

as contrast agents for ultra-sound (US) imaging. The water-gas interface created by the 

bubbles enhances existing images by reflecting sound waves more efficiently than tissue 

interfaces alone. Much interest has been gained recently to combine these advantages with 

other medical procedures, such as drug delivery or gene therapy. Conventionally, 

microbubble solutions are made by sonication or shaking of lipid solutions in a single step 

batch method. Whilst this provides adequate means to prepare simple microbubble 

solutions, if true multi-functional bubbles for imaging and therapy are to be realized, a more 

sophisticated generation method would be required. 

The area of microfluidics provides an exciting platform in which complex microbubbles can 

be generated in a controlled and reproducible manner by offering advancements in 

automation, fluid control, versatility and microbubble manipulation. Microbubbles are 

generated in flow-focussing microfluidic devices by nipping streams of gas and liquid 

through a tiny nozzle and then subjecting the two phases to a downstream pressure drop. To 

date microbubble generation has been performed solely on planar devices, in which 

pressure drops are created on a single axis. We propose a 3D expansion to further improve 

microbubble production rates by creating a large pressure drop in the outlet channel (figure 

1). In addition, we present a new bubble production regime. The microchip outlet expanded 

in a vertical direction from 25 µm at the nozzle to 50 µm in the outlet. Bubble concentrations 

produced using this device were up to 1 x 10 9 bubbles / mL, improving bubble 

concentrations from designs without the 3D expansion by a factor of 10. 

Figure 1. Photograph of the flow-focussing microfluidic device showing lipid 

and gas streams, the 3D expansion area and the bubble formation area.


4 th & 5 th July 2011 


Title: Counter flow microbubble channelling using acoustic radiation force funnel 

Benjamin Raiton 


Abstract: 

Background, Motivation and Objective 

The key advantage when employing acoustic travelling waves to manipulate microspheres is 

the ability to penetrate biological tissues. Other techniques such as optical tweezers or 

standing-wave traps are limited by their working distance. Previous publications reported on 

the use of Gaussian focused ultrasound to translate 125 μm lipid droplets and an opposite 

phase induced pressure black spot to align flowing particles. When microbubbles (MB) are 

used as drug carriers, a single element transducer is commonly used to emit a plane 

pressure wave. The effect, referred to as primary radiation force, is a translation of the MB 

away from the vessel lumen and towards the vessel wall to facilitate bonding to the 

endothelium. The axial primary radiation force (APRF) is calculated here according to Dayton 

et al. [1] along with the lateral primary radiation force (LPRF) following the simplified 

equation by Gor’kov [2]. 

Statement of Contribution/Methods 

Lipid MB used in the experiment have a size distribution of 1-10 μm. The MB are flowed 

through a 1 mm wide channel with a height of 100 μm, at a rate similar to that of venules. A 

low peak-negative-pressure (PNP) pulse of 200 cycles is emitted every 6.25 ms. The 96 

elements of a standard medical imaging probe are focused at the channel centre at 24 mm. 

Each half of the array transmits the same tone burst, but out of phase by 180°. 

Results 

In the figure, captured from beneath the vessel, the fluid is flowing from left to right. A clear 

formation of 3 parallel channels, flowing in the opposite direction, is observed as predicted 

by the simulation. The thin grey trails are made up of unaggregated MB whilst the larger and 

darker dots indicate MB clustering. The clusters break up as soon as the ultrasound source is 

switched off. 

Discussion and Conclusions 

Thanks to the use of a 1D linear array the spacing between the channels can be dynamically 

varied by altering the focal depth or number of elements used. The center of the three 

channels can also be shifted up and down by steering the focal point of the array. 

References: 

[1] P.A. Dayton et al., UFFC IEEE Trans, vol.44, no.6, pp,1264-1277, 1997 

*2+ L.P. Gor’kov, Sov. Phys. 6 (9), 773-775, 1962 


Benjamin Raiton, Sevan Harput, Dr James Mclaughlan, Dr Steven Freear 

Ultrasound Group, School of Electronic and Electrical Engineering, University of Leeds, Leeds, West Yorkshire, 

United Kingdom


4 th & 5 th July 2011 


Title: Comparision of methods for sizing and counting of microbubbles 

Charles Sennoga 

Imperial College London 

Abstract: The size and number count of microbubble populations are essential information 

for interpreting their acoustic behaviour and have a direct impact on clinical contrast 

enhanced ultrasound imaging and microbubble mediated therapy. There are three common 

ways of sizing and counting microbubbles, namely the electro-impedance volumetric zone 

sensing (ES) also called a Coulter Counter/Multisizer, optical microscopy (OM) and laser 

diffraction (LD). However the efficacy of these methods have not been thoroughly evaluated. 

In this study these methods was assessed. Microspheres with certified mean size diameter 

(MS) and number concentration (C) were used to assess sizing and counting reproducibility 

and reliability of ES, OM and LD methods. SonoVue was used to validate ES, OM and LD 

sizing and counting of microbubbles. Statistical analyses of intra-method variability for the 

SonoVue MS showed that the microbubble sizing was best obtained using OM compared 

to ES and LD. The microbubble counting reproducibility was best obtained using ES as 

compared to OM and LD. 


Charles A. Sennoga 12 , James S. M. Yeh 2 , Julia Alter 2 , Eleanor Stride 3 , Petros 

Nihoyannopoulos 4, John M. Seddon 5 , Dorian O. Haskard 4 , Joseph V. Hajnal 2 , Robert J. 

Eckersley 2 , Meng-Xing Tang 1 

1 Department of Bioengineering, Imperial College London, South Kensington Campus, London SW7 

2AZ, United Kingdom; 

2 Imaging Sciences Department, Imperial College London, Hammersmith Campus, London W12 0HS United 

Kingdom; 

3 Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE 

United Kingdom; 

4 National Heart and Lung Institute, Imperial College London, Hammersmith Campus, London W12 0HS United 

Kingdom; 

5 Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AY, United 

Kingdom;


4 th & 5 th July 2011 


Title: Pre-distorted chirp excitations for polydisperse microbubble populations 

Peter Smith 


Abstract: Ultrasound contrast agents are ideally monodisperse with single resonance frequency. 

Commercial microbubble populations however are often polydisperse e.g. SonoVue (approximately 

a 1-10 µm diameter range and mean value of 2.5 µm). As a consequence, a microbubble population 

will not be resonant at a single frequency but across a frequency range. The use of a frequency 

modulated or chirp excitation can increase the number of microbubbles which exhibit resonant 

behaviour within a population. However, a conventional chirp excitation will suffer from tapering 

effects according to the transducer’s bandwidth and frequency characteristics (as shown in the top 

plots of the figure). 

Whilst chirp excitation allows a large population of polydisperse microbubbles to be excited at their 

resonant frequencies; due to the nature of the transducer-tapered chirp, only a particular sub-set of 

the microbubble population are exposed to maximum pressure under resonant conditions. This work 

seeks to equalise the transmitted pressure across frequency so that all microbubbles within the 

population are subject to comparable pressure at their point of resonance. 

Chirp excitation sequences are designed which have an additional amplitude modulation function. 

This amplitude modulation pre-distorts the chirp signal in order to compensate for the filtering effect 

of the transducer. By pre-distorting the excitation sequence and shaping the pulse to reflect the 

attenuating response of the transducer, it is possible to achieve approximately constant pressure 

over a desired frequency band (as shown in the bottom plots of the figure). 

Excitation sequences are proposed that incorporate both the characteristics of the microbubble 

population and the frequency response of the transducer. Their aim is to achieve constant amplitude 

across a frequency range by effectively increasing the 3dB excitation bandwidth of the transducer. 

Additional authors: Peter R. Smith*, Dr David M. J. Cowell, Dr James McLaughlan and Dr Steven 

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


4 th & 5 th July 2011 

Delegate List 

Name Affiliation Email 

Mostafa Abdelrahman University of Leeds mtp5ma@leeds.ac.uk 

Radwa Abou-Saleh University of Leeds R.H.Saleh@leeds.ac.uk 

Muhammed Arif University of Leeds elma@leeds.ac.uk 

Jonathan Blockley University of Leeds pyjb@leeds.ac.uk 

Matthew Booth University of Leeds js06mab@leeds.ac.uk 

Christian Buchcic Wageningen University christian.buchcic@wur.nl 

Joanne Burke University of Leeds fs08jhb@leeds.ac.uk 

Richard Bushby University of Leeds R.J.Bushby@leeds.ac.uk 

Paul Campbell University of Dundee p.a.campbell@dundee.ac.uk 

Louise Coletta University of Leeds P.L.Coletta@leeds.ac.uk 

David Cowell University of Leeds D.M.J.Cowell@leeds.ac.uk 

Kevin Critchley University of Leeds K.Critchley@leeds.ac.uk 

Peter Culmer University of Leeds P.R.Culmer@leeds.ac.uk 

Verya Daeichin Erasmus MC v.daeichin@erasmusmc.nl 

Kari Dempsey Leeds General Infirmary kjd@medphysics.leeds.ac.uk 

Robert Eckersley Imperial College London. r.eckersley@csc.mrc.ac.uk 

Lydia Eidemueller VisualSonics leidemueller@visualsonics.com 

Stephen Evans University of Leeds S.D.Evans@leeds.ac.uk 

Tony Evans University of Leeds J.A.Evans@leeds.ac.uk 

Sarah Fawcett University of Cambridge Sarah.Fawcett@cancer.org.uk 

Damien Fouan Bf Systemes company damien.fouan@bf-systemes.fr 

Steve Freear University of Leeds S.Freear@leeds.ac.uk 

Caroline Harfield University College London c.harfield@ucl.ac.uk 

Sevan Harput University of Leeds eensha@leeds.ac.uk 

George Heath University of Leeds py06gh@leeds.ac.uk 

Carola Heneweer University Hospital Schleswig- 

Holstein, Kiel Germany 

c.heneweer@rad.uni-kiel.de 

Robert Hewson University of Leeds R.W.Hewson@leeds.ac.uk 

Nicola Ingram University of Leeds n.ingram@leeds.ac.uk 

Benjamin Johnson University of Leeds B.R.G.Johnson@leeds.ac.uk 

Pam Jones University of Leeds P.Jones@leeds.ac.uk 

Fabian Kiessling University of Aachen fkiessling@ukaachen.de 

Klazina Kooiman Erasmus MC k.kooiman@erasmusmc.nl


4 th & 5 th July 2011 

Dmitriy Kuvshinov University of Sheffield d.kuvshinov@sheffield.ac.uk 

Sean Lawler University of Leeds S.Lawler@leeds.ac.uk 

Gael Leaute University of Leeds elgyvl@leeds.ac.uk 

Ine Lentacker Ghent University Ine.Lentacker@UGent.be 

Marjorie Longo University of California mllongo@ucdavis.edu 

Mihaela Lorger University of Leeds medmlo@leeds.ac.uk 

Jonathan Loughran Imperial College London 

Paul Mackin Cambridge Research Institute Paul.Mackin@cancer.org.uk 

Sir Alex Markham University of Leeds A.F.Markham@leeds.ac.uk 

Gemma Marston University of Leeds G.Marston@leeds.ac.uk 

Jonathan McKendry University of Leeds jonomckendry@gmail.com 

James McLaughlan University of Leeds J.R.McLaughlan@leeds.ac.uk 

Gianluca Memoli National Physical Laboratory gianluca.memoli@npl.co.uk 

Serge Mensah CNRS mensah@lma.cnrs-mrs.fr 

Anne Neville University of Leeds A.Neville@leeds.ac.uk 

Richard O'Rorke University of Leeds R.D.O'Rorke@leeds.ac.uk 

Sally Peyman University of Leeds S.Peyman@leeds.ac.uk 

Malcolm Povey University of Leeds m.j.w.povey@food.leeds.ac.uk 

Ben Raiton University of Leeds elbdr@leeds.ac.uk 

Tijs Rovers Wageningen University tijs.rovers@wur.nl 

Tim Ryan Epigem Limited Tim.Ryan@epigem.co.uk 

Vassilis Sboros University of Edinburgh Vassilis.Sboros@ed.ac.uk 

Charles Sennoga Imperial College London c.sennoga@imperial.ac.uk 

Peter Smith University of Leeds efy3prs@leeds.ac.uk 

Jane Smith (was Bates) St James's University Hospital, Leeds Janes.Bates@leedsth.nhs.uk 

Eleanor Stride University College London eleanor.stride@ucl.ac.uk 

Chao Sun University of Edinburgh C.Sun-2@sms.ed.ac.uk 

Mengxing Tang Imperial College London mengxing.tang@imperial.ac.uk 

Neil Thomson University of Leeds N.H.Thomson@leeds.ac.uk 

Philippe Trochet VisualSonics ptrochet@visualsonics.com 

Richard Wakefield University of Leeds medrjw@leeds.ac.uk 

Nicholas Watson University of Leeds n.j.watson@leeds.ac.uk 

Stephen Wolstenhulme University of Leeds hcsswo@leeds.ac.uk

Microbubble Symposium: Fabrication, Characterisation and ...

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