In this study we present a combined optical sizing and acoustical characterization technique for the study of the dynamics of single freely-floating ultrasound contrast agent microbubbles exposed to long burst ultrasound excitations up to the milliseconds range. A co-axial flow device was used to position individual microbubbles on a streamline within the confocal region of three ultrasound
transducers and a high-resolution microscope objective. Bright-field images of microbubbles passing through the confocal region were captured using a high-speed camera synchronized to the acoustical data acquisition to assess the microbubble response to a 1-MHz ultrasound burst. Nonlinear bubble vibrations were identified at a driving pressure as low as 50 kPa. The results demonstrate good agreement with numerical simulations based on the shell-buckling model proposed by Marmottant et al. [J. Acoust. Soc. Am. 118, 3499–3505 (2005)]. The system demonstrates the potential for a high-throughput in vitro characterization of individual microbubbles.