Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 µm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

Functional ultrasound neuroimaging reveals mesoscopic organization of saccades in the lateral intraparietal area of posterior parietal cortex.

The lateral intraparietal cortex (LIP) located within the posterior parietal cortex (PPC) is an important area for the transformation of spatial information into accurate saccadic eye movements. Despite extensive research, we do not fully understand the functional anatomy of intended movement directions within LIP. This is in part due to technical challenges. Electrophysiology recordings can only record from small regions of the PPC, while fMRI and other whole-brain techniques lack sufficient spatiotemporal resolution. Here, we use functional ultrasound imaging (fUSI), an emerging technique with high sensitivity, large spatial coverage, and good spatial resolution, to determine how movement direction is encoded across PPC. We used fUSI to record local changes in cerebral blood volume in PPC as two monkeys performed memory-guided saccades to targets throughout their visual field. We then analyzed the distribution of preferred directional response fields within each coronal plane of PPC. Many subregions within LIP demonstrated strong directional tuning that was consistent across several months to years. These mesoscopic maps revealed a highly heterogenous organization within LIP with many small patches of neighboring cortex encoding different directions. LIP had a rough topography where anterior LIP represented more contralateral upward movements and posterior LIP represented more contralateral downward movements. These results address two fundamental gaps in our understanding of LIP's functional organization: the neighborhood organization of patches and the broader organization across LIP. These findings were achieved by tracking the same LIP populations across many months to years and developing mesoscopic maps of direction specificity previously unattainable with fMRI or electrophysiology methods.

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 in vivo to visualize cell location and activity or injected systemically to label and monitor tissue function. Distinguishing GVs from tissue signal deep inside intact organisms requires imaging approaches such as amplitude modulation (AM) or collapse-based pulse sequences, however they have limitations in sensitivity or require irreversible collapse of the GVs that restricts its scope for imaging dynamic cellular processes. To address these limitations, this study explores the utility of harmonic imaging to enhance the sensitivity of non-destructive imaging of GVs and cellular processes. Traditional fundamental-frequency imaging utilizing cross-wave AM (xAM) sequences has been deemed optimal for GV imaging. Contrary to this, we hypothesize that harmonic imaging, integrated with xAM could significantly elevate GV detection sensitivity. To verify our hypothesis, we conducted imaging on tissue-mimicking phantoms embedded with purified GVs, mammalian cells genetically modified to express GVs, and live mice after systemic GV infusion. Our findings reveal that harmonic xAM (HxAM) imaging markedly surpasses traditional xAM in isolating GVs' nonlinear acoustic signature, showcasing significant enhancements in signal-to-background and contrast-to-background ratios across all tested samples. Further investigation into the backscattered spectra elucidates the efficacy of harmonic imaging in conjunction with xAM. HxAM imaging enables the detection of lower concentrations of GVs and cells with ultrasound and extends the imaging depth in vivo by up to 20% and imaging performance metrics by up to 10dB. These advancements bolster the capabilities of ultrasound for molecular and cellular imaging, underscoring the potential of using harmonic signals to amplify GV detection.

SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression.

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed "stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes" (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper's transparent peer review process is included in the supplemental information.

Engineering viral vectors for acoustically targeted gene delivery.

Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific delivery of common gene vectors such as adeno-associated viruses (AAVs) is typically performed via invasive injections, which limit its applicable scope of research and clinical applications. Alternatively, focused ultrasound blood-brain-barrier opening (FUS-BBBO), performed noninvasively, enables the site-specific entry of AAVs into the brain from systemic circulation. However, when used in conjunction with natural AAV serotypes, this approach has limited transduction efficiency and results in substantial undesirable transduction of peripheral organs. Here, we use high throughput in vivo selection to engineer new AAV vectors specifically designed for local neuronal transduction at the site of FUS-BBBO. The resulting vectors substantially enhance ultrasound-targeted gene delivery and neuronal tropism while reducing peripheral transduction, providing a more than ten-fold improvement in targeting specificity in two tested mouse strains. In addition to enhancing the only known approach to noninvasively target gene delivery to specific brain regions, these results establish the ability of AAV vectors to be evolved for specific physical delivery mechanisms.

An implantable piezoelectric ultrasound stimulator (ImPULS) for deep brain activation.

Precise neurostimulation can revolutionize therapies for neurological disorders. Electrode-based stimulation devices face challenges in achieving precise and consistent targeting due to the immune response and the limited penetration of electrical fields. Ultrasound can aid in energy propagation, but transcranial ultrasound stimulation in the deep brain has limited spatial resolution caused by bone and tissue scattering. Here, we report an implantable piezoelectric ultrasound stimulator (ImPULS) that generates an ultrasonic focal pressure of 100 kPa to modulate the activity of neurons. ImPULS is a fully-encapsulated, flexible piezoelectric micromachined ultrasound transducer that incorporates a biocompatible piezoceramic, potassium sodium niobate [(K,Na)NbO3]. The absence of electrochemically active elements poses a new strategy for achieving long-term stability. We demonstrated that ImPULS can i) excite neurons in a mouse hippocampal slice ex vivo, ii) activate cells in the hippocampus of an anesthetized mouse to induce expression of activity-dependent gene c-Fos, and iii) stimulate dopaminergic neurons in the substantia nigra pars compacta to elicit time-locked modulation of nigrostriatal dopamine release. This work introduces a non-genetic ultrasound platform for spatially-localized neural stimulation and exploration of basic functions in the deep brain.

Functional ultrasound imaging of human brain activity through an acoustically transparent cranial window.

Visualization of human brain activity is crucial for understanding normal and aberrant brain function. Currently available neural activity recording methods are highly invasive, have low sensitivity, and cannot be conducted outside of an operating room. Functional ultrasound imaging (fUSI) is an emerging technique that offers sensitive, large-scale, high-resolution neural imaging; however, fUSI cannot be performed through the adult human skull. Here, we used a polymeric skull replacement material to create an acoustic window compatible with fUSI to monitor adult human brain activity in a single individual. Using an in vitro cerebrovascular phantom to mimic brain vasculature and an in vivo rodent cranial defect model, first, we evaluated the fUSI signal intensity and signal-to-noise ratio through polymethyl methacrylate (PMMA) cranial implants of different thicknesses or a titanium mesh implant. We found that rat brain neural activity could be recorded with high sensitivity through a PMMA implant using a dedicated fUSI pulse sequence. We then designed a custom ultrasound-transparent cranial window implant for an adult patient undergoing reconstructive skull surgery after traumatic brain injury. We showed that fUSI could record brain activity in an awake human outside of the operating room. In a video game "connect the dots" task, we demonstrated mapping and decoding of task-modulated cortical activity in this individual. In a guitar-strumming task, we mapped additional task-specific cortical responses. Our proof-of-principle study shows that fUSI can be used as a high-resolution (200 µm) functional imaging modality for measuring adult human brain activity through an acoustically transparent cranial window.

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 µm. 1 Within the past decade, reporter genes for US have been introduced 2,3 and engineered 4,5 to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). 6 Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semi-automated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologs of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

Truly Tiny Acoustic Biomolecules for Ultrasound Imaging and Therapy.

Nanotechnology offers significant advantages for medical imaging and therapy, including enhanced contrast and precision targeting. However, integrating these benefits into ultrasonography is challenging due to the size and stability constraints of conventional bubble-based agents. Here bicones, truly tiny acoustic contrast agents based on gas vesicles (GVs), a unique class of air-filled protein nanostructures naturally produced in buoyant microbes, are described. It is shown that these sub-80 nm particles can be effectively detected both in vitro and in vivo, infiltrate tumors via leaky vasculature, deliver potent mechanical effects through ultrasound-induced inertial cavitation, and are easily engineered for molecular targeting, prolonged circulation time, and payload conjugation.

Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures.

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nanocages, and other macromolecular assemblies, are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures' subcellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs' primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon reaction with the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. Importantly, this was achieved by replacing only a fraction of GvpA with the tagged version. We used fluorescence images of the tagged GVs to study the GV size and distance distributions within these cells. This bioorthogonal and fractional labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

Magneto-acoustic protein nanostructures for non-invasive imaging of tissue mechanics in vivo.

Measuring cellular and tissue mechanics inside intact living organisms is essential for interrogating the roles of force in physiological and disease processes. Current agents for studying the mechanobiology of intact, living organisms are limited by poor light penetration and material stability. Magnetomotive ultrasound is an emerging modality for real-time in vivo imaging of tissue mechanics. Nonetheless, it has poor sensitivity and spatiotemporal resolution. Here we describe magneto-gas vesicles (MGVs), protein nanostructures based on gas vesicles and magnetic nanoparticles that produce differential ultrasound signals in response to varying mechanical properties of surrounding tissues. These hybrid nanomaterials significantly improve signal strength and detection sensitivity. Furthermore, MGVs enable non-invasive, long-term and quantitative measurements of mechanical properties within three-dimensional tissues and in vivo fibrosis models. Using MGVs as novel contrast agents, we demonstrate their potential for non-invasive imaging of tissue elasticity, offering insights into mechanobiology and its application to disease diagnosis and treatment.

Decoding motor plans using a closed-loop ultrasonic brain-machine interface.

Brain-machine interfaces (BMIs) enable people living with chronic paralysis to control computers, robots and more with nothing but thought. Existing BMIs have trade-offs across invasiveness, performance, spatial coverage and spatiotemporal resolution. Functional ultrasound (fUS) neuroimaging is an emerging technology that balances these attributes and may complement existing BMI recording technologies. In this study, we use fUS to demonstrate a successful implementation of a closed-loop ultrasonic BMI. We streamed fUS data from the posterior parietal cortex of two rhesus macaque monkeys while they performed eye and hand movements. After training, the monkeys controlled up to eight movement directions using the BMI. We also developed a method for pretraining the BMI using data from previous sessions. This enabled immediate control on subsequent days, even those that occurred months apart, without requiring extensive recalibration. These findings establish the feasibility of ultrasonic BMIs, paving the way for a new class of less-invasive (epidural) interfaces that generalize across extended time periods and promise to restore function to people with neurological impairments.

Effects of focused ultrasound in a "clean" mouse model of ultrasonic neuromodulation.

Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3+/DTR × Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.

Bioorthogonal labeling enables in situ fluorescence imaging of expressed gas vesicle nanostructures.

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nano-cages, and other macromolecular assemblies are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures' sub-cellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs' primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon binding the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag, and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. We used fluorescence images of the tagged GVs to study GV size and distance distributions within these cells. This bioorthogonal labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

Using ultrasound to 3D-print materials.

Ultrasound allows additive manufacturing to go deeper-potentially inside the body.

Gas Vesicle-Blood Interactions Enhance Ultrasound Imaging Contrast.

Gas vesicles (GVs) are genetically encoded, air-filled protein nanostructures of broad interest for biomedical research and clinical applications, acting as imaging and therapeutic agents for ultrasound, magnetic resonance, and optical techniques. However, the biomedical applications of GVs as systemically injectable nanomaterials have been hindered by a lack of understanding of GVs' interactions with blood components, which can significantly impact in vivo behavior. Here, we investigate the dynamics of GVs in the bloodstream using a combination of ultrasound and optical imaging, surface functionalization, flow cytometry, and mass spectrometry. We find that erythrocytes and serum proteins bind to GVs and shape their acoustic response, circulation time, and immunogenicity. We show that by modifying the GV surface we can alter these interactions and thereby modify GVs' in vivo performance. These results provide critical insights for the development of GVs as agents for nanomedicine.

Ultrasonic reporters of calcium for deep tissue imaging of cellular signals.

Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standard fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultrasonic reporter of calcium (URoC). Based on a unique class of air-filled protein nanostructures called gas vesicles, we engineered URoC to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we demonstrate noninvasive ultrasound imaging of calcium signaling in vivo during drug-induced receptor activation. URoC brings the depth and resolution advantages of ultrasound to the in vivo imaging of dynamic cellular function and paves the way for acoustic biosensing of a broader variety of biological signals.

Living Material with Temperature-Dependent Light Absorption.

Engineered living materials (ELMs) exhibit desirable characteristics of the living component, including growth and repair, and responsiveness to external stimuli. Escherichia coli (E. coli) are a promising constituent of ELMs because they are very tractable to genetic engineering, produce heterologous proteins readily, and grow exponentially. However, seasonal variation in ambient temperature presents a challenge in deploying ELMs outside of a laboratory environment because E. coli growth rate is impaired both below and above 37 °C. Here, a genetic circuit is developed that controls the expression of a light-absorptive chromophore in response to changes in temperature. It is demonstrated that at temperatures below 36 °C, the engineered E. coli increase in pigmentation, causing an increase in sample temperature and growth rate above non-pigmented counterparts in a model planar ELM. On the other hand, at above 36 °C, they decrease in pigmentation, protecting the growth compared to bacteria with temperature-independent high pigmentation. Integrating the temperature-responsive circuit into an ELM has the potential to improve living material performance by optimizing growth and protein production in the face of seasonal temperature changes.

Remote control of mechanochemical reactions under physiological conditions using biocompatible focused ultrasound.

External control of chemical reactions in biological settings with spatial and temporal precision is a grand challenge for noninvasive diagnostic and therapeutic applications. While light is a conventional stimulus for remote chemical activation, its penetration is severely attenuated in tissues, which limits biological applicability. On the other hand, ultrasound is a biocompatible remote energy source that is highly penetrant and offers a wide range of functional tunability. Coupling ultrasound to the activation of specific chemical reactions under physiological conditions, however, remains a challenge. Here, we describe a synergistic platform that couples the selective mechanochemical activation of mechanophore-functionalized polymers with biocompatible focused ultrasound (FUS) by leveraging pressure-sensitive gas vesicles (GVs) as acousto-mechanical transducers. The power of this approach is illustrated through the mechanically triggered release of covalently bound fluorogenic and therapeutic cargo molecules from polymers containing a masked 2-furylcarbinol mechanophore. Molecular release occurs selectively in the presence of GVs upon exposure to FUS under physiological conditions. These results showcase the viability of this system for enabling remote control of specific mechanochemical reactions with spatiotemporal precision in biologically relevant settings and demonstrate the translational potential of polymer mechanochemistry.

Location-aware ingestible microdevices for wireless monitoring of gastrointestinal dynamics.

Localization and tracking of ingestible microdevices in the gastrointestinal (GI) tract is valuable for the diagnosis and treatment of GI disorders. Such systems require a large field-of-view of tracking, high spatiotemporal resolution, wirelessly operated microdevices and a non-obstructive field generator that is safe to use in practical settings. However, the capabilities of current systems remain limited. Here, we report three dimensional (3D) localization and tracking of wireless ingestible microdevices in the GI tract of large animals in real time and with millimetre-scale resolution. This is achieved by generating 3D magnetic field gradients in the GI field-of-view using high-efficiency planar electromagnetic coils that encode each spatial point with a distinct magnetic field magnitude. The field magnitude is measured and transmitted by the miniaturized, low-power and wireless microdevices to decode their location as they travel through the GI tract. This system could be useful for quantitative assessment of the GI transit-time, precision targeting of therapeutic interventions and minimally invasive procedures.