The microcirculation can be differentiated from the surrounding tissue using high frequency ultrasound subharmonic imaging. This imaging technique relies on the detection of energy scattered from ultrasound contrast agents at half the transmit frequency due to their resonant oscillations. The current contrast agents and the subharmonic imaging parameters have not been optimized for high frequencies. Moreover, the origin of subharmonic generation from submicron bubbles is not well-understood. The size distribution of Definity™ phospholipid-shelled microbubbles was altered to find the optimal bubble size to be resonant over a wide range of high frequencies. The resonant behaviour of bubbles was investigated through in vitro attenuation measurements. The transmit frequency and pressure were varied to optimize the backscattered subharmonic signal. Alteration of Definity™ population significanatly improved the scattering for subharmonic imaging at 20 MHz. A peak negative pressure between 400 to 600 kPa is suggested for this frequency range.