Harmonic imaging for nonlinear detection of acoustic biomolecules.

Gas vesicles (GVs) based on acoustic reporter genes have emerged as potent contrast agents for cellular and molecular ultrasound imaging. These air-filled, genetically encoded protein nanostructures can be expressed in a variety of cell types to visualize cell location and activity or injected systemically to label and monitor tissue function. Distinguishing GV signal from tissue deep inside intact organisms requires imaging approaches such as amplitude modulation (AM) or collapse-based pulse sequences. However, these approaches have limitations either in sensitivity or require the destruction of GVs, restricting the imaging of dynamic cellular processes. To address these limitations, we developed harmonic imaging to enhance the sensitivity of nondestructive GV imaging. We hypothesized that harmonic imaging, integrated with AM, could significantly elevate GV detection sensitivity by leveraging the nonlinear acoustic response of GVs. We tested this hypothesis by imaging tissue-mimicking phantoms embedded with purified GVs, mammalian cells genetically modified to express GVs, and mice liver post-systemic infusion of GVs. Our findings reveal that harmonic cross-propagating wave AM (HxAM) imaging markedly surpasses traditional xAM in isolating GVs' nonlinear acoustic signature, demonstrating significant (p < 0.05) enhancements in imaging performance. HxAM imaging improves detection of GV producing cells up to three folds , enhances imaging performance by over 10 dB, while extending imaging depth by up to 20%. Investigation into the backscattered spectra further elucidates the advantages of harmonic imaging. These advancements bolster ultrasound's capability in molecular and cellular imaging, underscoring the potential of harmonic signals to improve GV detection.