Hadamard-Encoded Pulses with Amplitude Modulation for Contrast-Enhanced Ultrasound Imaging of Acoustic Bacteria

Genetically engineered bacteria expressing gas vesicles (GVs), i.e., acoustic bacteria, enhance ultrasound contrast by leveraging their gas-filled nanostructures, playing a significant role in disease detection and assisting therapeutic interventions by visualizing intratumoral bacterial colonization and distribution. However, in vivo imaging with acoustic bacteria suffers from tissue background noise interference, which critically limits contrast enhancement. To address this challenge, we developed a nonlinear imaging paradigm integrating Hadamard-encoded pulse sequences with amplitude modulation (Hada-AM), complemented with a fundamental frequency filter. Through phantom and in vivo experiments, we demonstrated that the proposed Hada-AM method substantially outperforms conventional contrast-enhanced ultrasound (CEUS) imaging techniques in enhancing the contrast of acoustic bacterial signals, providing a new acoustic visualization strategy for engineered bacteria for use in tumor diagnosis and therapy.