In the previous grant period, experiments were performed to develop an understanding of the bubble generation process in the urinary tract and our knowledge of the acoustic field requirements for contrast production has greatly improved. Excise canine urinary bladders and initial in vivo trails demonstrated that single acoustic bursts of 6.6 Mpa and 250 ms duration produced, in a few in vitro and in vivo cases, clouds of bubbles with apparently quite sufficient echogenicity and duration for reflux diagnosis. However, such bursts, even up to 14 Mpa, would not reliably produce noticeable cavitation under these conditions where both the particulate and gas content is low. However, the threshold was lower (8.9 +/- 1.7 Mpa with 125 ms bursts) and reliability of bubble generation was significantly greater (7 or 8 animals) in a rabbit model where naturally occurring particulates may have reduced the threshold to an obtainable level with the employed pulsed system. This allowed a study of the effects of natural gas content variation on echogenicity yield which indicated that copious quantities of cavitation could be produced in vivo and that CO2 content was important in threshold and echogenicity. Controlled experiments in water were also performed, which again indicated that under conditions where threshold could be reached reliably, physiologically relevant gas contents could produce copious bubble production. The implication is that with sufficient acoustic amplitude to reach the cavitation threshold, bubbles can be produced in fluids with gas contents similar to that of urine. Recent canine experiments indicate that the goal of reliable generation has now been achieved using a short pulse system with up to twice the amplitude and with pulse sequences on only 1/4000 as much on-time as the earlier 250 ms pulses. Cavitation was repeatedly produced in the canine bladder with apparently no gross damage to the bladder wall. While the amount of echogenicity was lower than that found for the rabbit model, the repeated pulsing which can be performed with this system, or a low amplitude growth pulse should produce the requisite echogenicity and bubble duration, particularly if physiologic manipulation of CO2 in the urine is sufficiently safe for cases of low gas content. Given this level of success, it is the objective of the research proposed to develop a system to generate, collect and grow microbubbles to the degree necessary for diagnosis of urinary reflux. To this end, the proposed research focuses on low particle content systems where the effects of pulse parameters affecting safety, and contrast longevity and detectability can be addressed to develop a working in vivo system.