Ultrasound contrast agents (UCA) populations are typically polydisperse and contain microbubbles with radii over a given range. Although the behavior of microbubbles of certain sizes might be masked by the behavior of others, the acoustic characterization of UCA is typically made on full populations. In this paper, we have combined acoustic and optical methods to investigate the response of isolated lipid-shelled microbubbles to low-pressure (49 and 62 kPa peak negative pressure) ultrasound tone bursts. These bursts induced slow deflation of the microbubbles. The experimental setup included a microscope connected to a fast camera acquiring one frame per pulse transmitted by a single-element transducer. The behavior of each bubble was measured at multiple frequencies, by cyclically changing the transmission frequency over the range of 2 to 4 MHz during subsequent pulse repetition intervals. The bubble echoes were captured by a second transducer and coherently recorded. More than 50 individual microbubbles were observed. Microbubbles with radii larger than 3 mum did not experience any size reduction. Smaller bubbles slowly deflated, generally until the radius reached a value around 1.4 microm, independent of the initial microbubble size. The detected pressure amplitude backscattered at 2.5 cm distance was very low, decreasing from about 5 Pa down to 1 Pa at 2 MHz as the bubbles deflated. The resonant radius was evaluated from the echo amplitude normalized with respect to the geometrical cross section. At 2-MHz excitation, deflating microbubbles showed highest normalized echo when the radius was 2.2 microm while at higher excitation frequencies, the resonant radius was lower. The relative phase shift of the echo during the deflation process was also measured. It was found to exceed pi/2 in all cases. A heuristic procedure based on the analysis of multiple bubbles of a same population was used to estimate the undamped natural frequency. It was found that a microbubble of 1.7 microm has an undamped natural frequency of 2 MHz. The difference between this size and the resonant radius is discussed as indicative of significant damping.