Cross amplitude modulation imaging: theory and basic principles

The introduction of genetically encoded gas vesicles (GVs), protein nanostructures with the ability to scatter sound, has created the possibility for deep tissue cellular imaging. GVs establish a platform for biomolecular engineering and were successfully repurposed into acoustic reporter genes and acoustic biosensors. Alongside molecular engineering developments, a method called cross amplitude modulation (xAM) has emerged as the gold standard for non-destructive ultrasound imaging of GVs thanks to its sensitivity and specificity in living biological tissues. Here, we present latest xAM theory and imaging principles. Specifically, we report 1) analytical expressions for the X-wave beam width and primary-to-secondary lobes distance; 2) experimental observations of nondiffractive xAM beams; 3) a method to modulate the secondary lobe level of xAM beams; 4) a demonstration of the incoherent nature of the xAM image noise that can be leverage to increase sensitivity through frame averaging, 5) a beamforming formalism to enhance xAM contrast-to-noise ratio without reducing framerate. Ultimately, the rise of the field of Biomolecular Ultrasound will rest on the co-development of genetically encoded probes and dedicated imaging methods such as xAM and its 3D extension, nonlinear sound-sheet microscopy.