Investigation of the response of attached biSphere (TM) microbubbles to ultrasound

Knowledge of the behaviour of individual contrast microbubbles is essential if ultrasound contrast agents are to be developed to their full potential. In order to investigate the acoustic response of attached microbubbles an experimental system has been developed that can be used for sequential optical and acoustical imaging of attached particles. The aim of the work described here was to determine the feasibility of using the system for the study of single attached microbubbles.

The experimental system, comprising tank and sliding device was mounted on a Leica inverted microscope alongside a Philips Sonos 5500 scanner and S3 transducer. The beam axis was positioned at 80 degrees to the surface of a 12 mu m thick polyester membrane. Single Copper (Cu) spheres were attached to the membrane and used to align the acoustic and optical fields. BiSphere (TM) microbubbles (Point Biomedical) were treated to fluoresce and attached to the membrane using poly-L-lysine and an inversion technique. On one section of the membrane a patch (similar to 1 mm diameter) of densely packed bubbles was used to confirm the alignment. In other areas on the membrane a diluted solution of biSphere (TM) was attached which allowed one microbubble per field of view to be isolated. At x10 magnification the field of view was approximately 2 mm, with the ultrasound beamwidth at the area of interest being 4mm. In order to determine the response of attached biSphere (TM), a range of acoustic pressures upto 1000 kPa and frequencies of 1.57 and 3 MHz were used to insonate patches and single bubbles of biSphere (TM) A 6 cycle pulse was used, and unprocessed backscattered RF data was captured.

For patches of biSphere (TM), the backscatter signal was seen to remain constant with time at low acoustic pressures (400 kPa) while at acoustic pressures up to 1000 kPa an acoustic signal was observed that was consistent with gas escaping from attached bubbles. The fluorescent shells remained attached to the membrane after gas escape. For single attached biSphere (TM) bubbles, no acoustic signal was detected at low acoustic pressures and at high acoustic pressures an acoustic signal from the escaping gas was recorded. It will be possible to modify the experimental system to allow the study of the transfer of materials from microbubbles to cells.