Visualizing and modulating molecular and cellular processes occurring deep within living organisms is fundamental to our study of basic biology and disease. Currently, the most sophisticated tools available to dynamically monitor and control cellular events rely on light-responsive proteins, which are difficult to use outside of optically transparent model systems, cultured cells, or surgically accessed regions owing to strong scattering of light by biological tissue. In contrast, ultrasound is a widely used medical imaging and therapeutic modality that enables the observation and perturbation of internal anatomy and physiology but has historically had limited ability to monitor and control specific cellular processes. Recent advances are beginning to address this limitation through the development of biomolecular tools that allow ultrasound to connect directly to cellular functions such as gene expression. Driven by the discovery and engineering of new contrast agents, reporter genes, and bioswitches, the nascent field of biomolecular ultrasound carries a wave of exciting opportunities.

Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging.

Most cellular phenomena of interest to mammalian biology occur within the context of living tissues and organisms. However, today’s most advanced tools for observing and manipulating cellular function – based on fluorescent or light-controlled proteins – work best in cultured cells, transparent model species or small, surgically accessed anatomical regions. Their reach into deep tissues and larger animals is limited by photon scattering. To overcome this limitation, we must design biochemical tools that interface with more penetrant forms of energy. For example, sound waves and magnetic fields easily permeate most biological tissues, allowing the formation of images and delivery of energy for actuation. These capabilities are widely used in clinical techniques such as diagnostic ultrasound, magnetic resonance imaging, focused ultrasound ablation and magnetic particle hyperthermia. Each of these modalities offers spatial and temporal precision that could be used to study a multitude of cellular processes in vivo. However, connecting these techniques to cellular functions such as gene expression, proliferation, migration and signaling requires the development of new biochemical tools that can interact with sound waves and magnetic fields as optogenetic tools interact with photons. Here, we discuss the exciting challenges this poses for biomolecular engineering, and provide examples of recent advances pointing the way to greater depth in in vivo cell biology.

The ability to physically manipulate specific cells is critical for the fields of biomedicine, synthetic biology, and living materials. Ultrasound has the ability to manipulate cells with high spatiotemporal precision via acoustic radiation force (ARF). However, because most cells have similar acoustic properties, this capability is disconnected from cellular genetic programs. Here, we show that gas vesicles (GVs)—a unique class of gas-filled protein nanostructures—can serve as genetically encodable actuators for selective acoustic manipulation. Because of their lower density and higher compressibility relative to water, GVs experience strong ARF with opposite polarity to most other materials. When expressed inside cells, GVs invert the cells’ acoustic contrast and amplify the magnitude of their ARF, allowing the cells to be selectively manipulated with sound waves based on their genotype. GVs provide a direct link between gene expression and acoustomechanical actuation, opening a paradigm for selective cellular control in a broad range of contexts.

Ultrasound is playing an emerging role in molecular and cellular imaging thanks to new micro-and nanoscale contrast agents and reporter genes. Acoustic methods for the selective in vivo detection of these imaging agents are needed to maximize their impact in biology and medicine. Existing ultrasound pulse sequences use the nonlinearity in contrast agents' response to acoustic pressure to distinguish them from mostly linear tissue scattering. However, such pulse sequences typically scan the sample using focused transmissions, resulting in a limited frame rate and restricted field of view. Meanwhile, existing wide-field scanning techniques based on plane wave transmissions suffer from limited sensitivity or nonlinear artifacts. To overcome these limitations, we introduce an ultrafast nonlinear imaging modality combining amplitude-modulated pulses, multiplane wave transmissions and selective coherent compounding. This technique achieves contrast imaging sensitivity comparable to much slower gold-standard amplitude modulation sequences and enables the acquisition of larger and deeper fields of view, while providing a much faster imaging framerate of 3.2kHz. Additionally, it enables simultaneous nonlinear and linear image formation, and allows concurrent monitoring of phenomena accessible only at ultrafast framerates, such as blood volume variations. We demonstrate the performance of this ultrafast amplitude modulation (uAM) technique by imaging gas vesicles, an emerging class of genetically encodable biomolecular contrast agents, in several in vitro and in vivo contexts. These demonstrations include the rapid discrimination of moving contrast agents and the real-time monitoring of phagolysosomal function in the mouse liver.