Relationships between scattered signals from ultrasonically activated contrast agents and cell membrane damage in vitro

Phagocytic cells of a mouse macrophage cell line growing on thin Mylar sheets were exposed to ultrasound in the presence of an ultrasound contrast agent (Optison(R)). Most of the gas microbubbles (i.e. gas bodies) of the contrast agent remain attached to the cells even after rinsing, which make the cells targets for bioeffects. The cell monolayers were incubated in a chamber with 2% of Optison(R) for 15 minutes before rinsing. Ultrasound exposures were made at a frequency of 2.25 MHz with 46 cycle pulses and a PRF of 1 kHz. The ultrasound exposures had different numbers of pulses (1, 2, 3, and 10). Each was used with four peak negative pressures (0.5, 1, 2, and 3 MPa). The scattered signal from the monolayer containing cells and contrast agents was sensed by a 1 MHz receiver transducer and captured using a high speed digitizing board. The spectrum for the central received pulse was then computed and normalized to a control spectrum from linear scatterers.. After the exposure, a photomicrograph was made within the area of the monolayer exposed to the ultrasound beam, and the percentage of blue stained cells, indicative of cell membrane damage, was determined. Most of cell membrane damage occurs within the first 3 pulses. The Relative Integrated Power Spectrum (RIPS) of the subharmonic and ultraharmonics (3/2, 5/2, 7/2 and 9/2) show a sharp variation within the first 3-5 pulses. The cumulative function of the RIPS of the subharmonic seems to be a good predictive parameter for cell membrane damage. The measurement of scattered sub- and ultra harmonics could be useful for remotely sensing the location and magnitude of cavitation activity and non thermal bioeffects during diagnostic or therapeutic applications.