Ultrasound imaging is a cornerstone of medical diagnostics, offering high-resolution, real-time visualization of anatomical structures. However, its application to molecular and cellular imaging has been limited by the lack of nanoscale contrast agents. Gas vesicles (GVs), air-filled protein nanostructures evolved for buoyancy in microorganisms, offer transformative potential in this domain. This thesis explores the engineering and application of GVs as biomolecular ultrasound contrast agents, emphasizing their use as biosensors for molecular imaging. It builds on their unique genetic encodability, tunable acoustic properties, and nanoscale dimensions to address key limitations in ultrasound imaging.

In chapter 1 and chapter 2 we introduce the potential of GVs as genetically encoded ultrasound contrast agents, highlighting their advantages over traditional agents like microbubbles. These chapters provide an overview of ultrasound imaging’s evolution, focusing on the emerging field of biomolecular ultrasound imaging, which leverages GVs to bridge the gap between molecular processes and ultrasound modalities. Applications such as neuroscience imaging and functional imaging of dynamic biological processes are discussed, emphasizing GVs’ nanoscale properties and unique acoustic behavior.

The contents of chapter 3 focus on the cryo-electron microscopy (cryo-EM) structural analysis of GVs. This study provides an atomic-level model of the GV shell, particularly in the absence of the reinforcement protein GvpC. By combining structural insights with a sequence analysis of GvpC, the chapter proposes a hypothetical binding mechanism that informs mutagenesis experiments in later work. This structural foundation is critical for the subsequent engineering of GVs for biosensor applications.

In chapter 4 we present the development and validation of pHonon, the first GV-based pH biosensor. By engineering pH-sensitive histidine residues into GvpC, the biosensor’s acoustic properties were tuned to detect pH variations. Validation experiments, conducted both in vitro and in vivo, demonstrated pHonon’s efficacy for real-time, non-invasive pH imaging. This work highlights the versatility of GVs as platforms for biosensor engineering and their potential for applications in both basic research and clinical diagnostics.

Then, chapter 5 explores alternative approaches to enhancing GV functionality through aggregation. By inducing GV clustering via methods like biotin-streptavidin interactions and depletion interactions, significant improvements in ultrasound contrast were achieved. This chapter shows that aggregation enhances both linear and non-linear acoustic responses, providing a complementary strategy to genetic engineering for optimizing GV performance. These findings open new avenues for improving the signal strength and utility of GVs in various imaging applications.

The thesis concludes by summarizing the key findings and their implications for the field of biomolecular ultrasound imaging. It emphasizes the breakthroughs achieved, such as the high-resolution GV structural model, the development of pHonon, and the exploration of aggregation-based contrast enhancement. These contributions advance the field significantly, offering innovative solutions to challenges in molecular imaging. This thesis lays the basis for future research on broadening the array of biomarkers identifiable by GVs, improving genetic engineering methods, and investigating additional imaging techniques to enhance the effectiveness of biomolecular ultrasound imaging.

Light-sheet fluorescence microscopy has revolutionized biology by visualizing dynamic cellular processes in three dimensions. However, light scattering in thick tissue and photobleaching of fluorescent reporters limit this method to studying thin or translucent specimens. In this study, we applied nondiffractive ultrasound beams in conjunction with a cross-amplitude modulation sequence and nonlinear acoustic reporters to enable fast and volumetric imaging of targeted biological functions. We reported volumetric imaging of tumor gene expression at the cubic centimeter scale using genetically encoded gas vesicles and localization microscopy of cerebral capillary networks using intravascular microbubble contrast agents. Nonlinear sound-sheet microscopy provides a ~64× acceleration in imaging speed, ~35× increase in imaged volume, and ~4× increase in classical imaging resolution compared with the state of the art in biomolecular ultrasound.

Gas vesicles are gas-filled nanocompartments that allow a diverse group of bacteria and archaea to control their buoyancy. The molecular basis of their properties and assembly remains unclear. Here, we report the 3.2 Å cryo-EM structure of the gas vesicle shell made from the structural protein GvpA that self-assembles into hollow helical cylinders closed off by cone-shaped tips. Two helical half shells connect through a characteristic arrangement of GvpA monomers, suggesting a mechanism of gas vesicle biogenesis. The fold of GvpA features a corrugated wall structure typical for force-bearing thin-walled cylinders. Small pores enable gas molecules to diffuse across the shell, while the exceptionally hydrophobic interior surface effectively repels water. Comparative structural analysis confirms the evolutionary conservation of gas vesicle assemblies and demonstrates molecular features of shell reinforcement by GvpC. Our findings will further research into gas vesicle biology and facilitate molecular engineering of gas vesicles for ultrasound imaging.

Ultrasound imaging is one of the most widely used modalities in clinical practice, revealing human prenatal development but also arterial function in the adult brain. Ultrasound waves travel deep within soft biological tissues and provide information about the motion and mechanical properties of internal organs. A drawback of ultrasound imaging is its limited ability to detect molecular targets due to a lack of cell-type specific acoustic contrast. To date, this limitation has been addressed by targeting synthetic ultrasound contrast agents to molecular targets. This molecular ultrasound imaging approach has proved to be successful but is restricted to the vascular space. Here, we introduce the nascent field of biomolecular ultrasound imaging, a molecular imaging approach that relies on genetically encoded acoustic biomolecules to interface ultrasound waves with cellular processes. We review ultrasound imaging applications bridging wave physics and chemical engineering with potential for deep brain imaging.