![]() ![]() Amplitude modulation (AM) ultrasound pulse sequences emerged as logical candidates to exploit dissimilar hGV responses below and above buckling and enabled nonlinear imaging of hGVs in vitro, in ovo, and in vivo. engineered harmonic GV variants (hGVs) that buckle and scatter higher harmonics at acoustic pressures above 320 kPa, corresponding to a mechanical index of 0.08, well below the FDA safety requirement of 1.9. While most natural GVs behave as linear ultrasound scatters, Lakshmanan et al. In 2014, it was reported that GVs could be imaged with ultrasound, and it was recently shown that gene clusters encoding GVs could be expressed heterologously in engineered cells and serve as acoustic reporter genes. GVs are air-filled nanocompartments with dimensions on the order of 200 nm, enclosed by a rigid 2 nm-thick protein shell. To maximize the impact of acoustic biomolecules in biology and medicine, physical methods are needed to discriminate GV scattering from tissue scattering, analogous to previous developments in the imaging of synthetic microbubble contrast agents. Very recently, the first acoustic biomolecules for ultrasound, analogous to GFP for optics, were developed based on a unique class of air-filled protein nanostructures called gas vesicles, or GVs, making it possible to use ultrasound to visualize the function of cells deep inside tissues. In contrast, the physics of ultrasonic waves allow them to propagate centimeters deep into biological tissues without losing their coherence, and enable tissue scanning at the organ scale. However, the microstructure of biological tissues restricts a photon’s transport mean free path to about 1 mm, limiting in vivo optical imaging applications. ![]() Green fluorescent protein (GFP) and its analogs serve as irreplaceable tools allowing biologists to visualize gene expression and other cellular processes using optical microscopes. These results advance the physical facet of the emerging field of biomolecular ultrasound, and are also relevant to synthetic ultrasound contrast agents. Finally, we demonstrate that xAM enables highly specific in vivo imaging of GVs located in the gastrointestinal tract, a target of prime interest for future cellular imaging. We demonstrate in tissue-mimicking phantoms that imaging artifacts distal to GV inclusions decrease as the plane-wave cross-propagation angle opens, nearing complete extinction at angles above 16.5 degrees. We show in both simulations and experiments that residual xAM nonlinearity due to wave propagation decreases as the plane-wave cross-propagation angle increases. By transmitting cross-propagating plane-waves, we minimize cumulative nonlinear interaction effects due to collinear wave propagation, while generating a transient wave-amplitude modulation at the two plane-waves’ intersection. The xAM method derives from counter-propagating wave interaction theory which predicts that, in media exhibiting quadratic elastic nonlinearity like biological tissue, the nonlinear interaction of counter-propagating acoustic waves is inefficient. To address this issue, we present an imaging paradigm, cross-amplitude modulation (xAM), which relies on cross-propagating plane-wave transmissions of finite aperture X-waves to achieve quasi artifact-free in vivo imaging of GVs. Unfortunately, the in vivo specificity of AM ultrasound imaging is systematically compromised by the nonlinearity added by the GVs to propagating waves, resulting in strong image artifacts from linear scatterers downstream of GV inclusions. We previously engineered GVs exhibiting a nonlinear scattering behavior in response to acoustic pressures above 300 kPa, and showed that amplitude-modulated (AM) ultrasound pulse sequences that both excite the linear and nonlinear GV scattering regimes were highly effective at distinguishing GVs from linear scatterers like soft biological tissues. Acoustical methods for the in vivo detection of GVs are now required to maximize the impact of this technology in biology and medicine. ![]() The expression of these unique air-filled proteins, known as gas vesicles (GVs), in cells allows ultrasound to image cellular functions such as gene expression in vivo, providing ultrasound with its analog of optical fluorescent proteins. Recently, this capability was enhanced with the development of acoustic biomolecules – proteins with physical properties enabling them to scatter sound. ![]() The basic physics of sound waves enables ultrasound to visualize biological tissues with high spatial and temporal resolution. ![]()
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