Development of a novel experimental set-up to allow investigation of the ultrasonic backscatter from microbubble contrast agents attached to surfaces

Developing applications of ultrasound contrast agents include targeting to areas such as inflamed plaque, drug delivery and gene therapy. In order to develop these techniques to their full potential the interaction of individual attached microbubbles with ultrasound and the mechanisms of transfer of material from microbubble to targeted areas needs to be fully understood.

Aim: To develop an experimental set-up and technique suitable for determining the ultrasonic back-scatter from individual, attached microbubbles and for the study of the transfer of material from single bubbles.

A tank allowing acoustic and optical imaging was constructed with dimensions of: optical imaging section 34 x 16 x 4 cm, acoustic imaging section 30 x 16 x 30 cm. A microscope slide incorporated into the base of the optical imaging section maximised the quality of the microscope images. The imaging modalities were separate in the tank to allow precise characterisation of the acoustic field and to minimise reflections from surfaces other than the membrane. A holder comprising two Perspex rings held a 12 mu m polyester membrane. A sliding device allowed movement of the membrane holder between the ultrasound field and the microscope. A membrane hydrophone with active element of 0.5 mm was used to determine the acoustic field at the surface of the membrane and within the tank. Copper (Cu) spheres of diameter 40-80 mu m, attached to the membrane using poly-L-lysine (PLL), were used to assess the experimental set-up and to aid with alignment of the microscope optics with the acoustic field. A Sonos5500 scanner with S3 transducer of frequency range 1.26-3.75 MHz was used. The transducer was placed at an angle to the membrane to minimise the received echo, the area of interest on the membrane was 7.5 cm from the transducer. A sequence of 6 pulses was used. Previously the minimum detectable pressure with the ultrasound settings was determined to be 0.1 Pa. Cu spheres were used to assess the suitability of the set-up for studying microbubbles. Commercially available Definity microbubbles, mean diameter 1.1-3.3 mu m, have been attached using PLL and imaged with 2 MHz ultrasound.

The acoustic field was characterised for 1.26-3.75 MHz, for acoustic pressures of 300 and 550 kPa. Using the sliding device, Cu spheres attached to the membrane were found to reposition over the microscope objective within 20 pin of the original position which will allow optical imaging of contrast agents before and after insonation.

The tank and set up has been shown to allow detection of the ultrasonic backscatter from individual particles attached to a membrane where the ultrasonic field at the location of the particle can be well calibrated. Definity attached to a membrane can be imaged and future experiments will investigate ultrasonic backscatter from attached single microbubbles for frequencies up to 11.1 MHz.