AbstractRecent advances have demonstrated the feasibility of molecular imaging using targeted microbubbles and ultrasound. One technical challenge is to selectively detect attached bubbles from those freely flowing bubbles and surrounding tissue. Pulse Inversion Doppler is an imaging technique enabling the selective detection of both static and moving ultrasound contrast agents: linear scatterers generate a single band Doppler spectrum, while non-linear scatterers generate a double band spectrum, one being uniquely correlated with the presence of contrast agents and non-linear tissue signals. We demonstrate that similar spectrums, and thus the same discrimination, can be obtained through a Doppler implementation of Pulse Subtraction. This is achieved by reconstructing a virtual echo using the echo generated from a short pulse transmission. Moreover by subtracting from this virtual echo the one generated from a longer pulse transmission, it is possible to fully suppress the echo from linear scatterers, while for non-linear scatterers, a signal will remain, allowing classical agent detection. Simulations of a single moving microbubble and a moving linear scatterer subject to these pulses show that when the virtual echo and the long pulse echo are used to perform pulsed Doppler, the power Doppler spectrum allows separation of linear and non-linear moving scattering. Similar results are obtained on experimental data acquired on a flow containing either microbubble contrast agents or linear blood mimicking fluid. This new Doppler method constitutes an alternative to Pulse Inversion Doppler and preliminary results suggest that similar dual band spectrums could be obtained by the combination of any non-linear detection technique with Doppler demodulation.