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.

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.

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.

Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria.

Ultrasound allows imaging at a much greater depth than optical methods, but existing genetically encoded acoustic reporters for in vivo cellular imaging have been limited by poor sensitivity, specificity and in vivo expression. Here we describe two acoustic reporter genes (ARGs)-one for use in bacteria and one for use in mammalian cells-identified through a phylogenetic screen of candidate gas vesicle gene clusters from diverse bacteria and archaea that provide stronger ultrasound contrast, produce non-linear signals distinguishable from background tissue and have stable long-term expression. Compared to their first-generation counterparts, these improved bacterial and mammalian ARGs produce 9-fold and 38-fold stronger non-linear contrast, respectively. Using these new ARGs, we non-invasively imaged in situ tumor colonization and gene expression in tumor-homing therapeutic bacteria, tracked the progression of tumor gene expression and growth in a mouse model of breast cancer, and performed gene-expression-guided needle biopsies of a genetically mosaic tumor, demonstrating non-invasive access to dynamic biological processes at centimeter depth.

Geometric effects in gas vesicle buckling under ultrasound.

Acoustic reporter genes based on gas vesicles (GVs) have enabled the use of ultrasound to noninvasively visualize cellular function in vivo. The specific detection of GV signals relative to background acoustic scattering in tissues is facilitated by nonlinear ultrasound imaging techniques taking advantage of the sonomechanical buckling of GVs. However, the effect of geometry on the buckling behavior of GVs under exposure to ultrasound has not been studied. To understand such geometric effects, we developed computational models of GVs of various lengths and diameters and used finite element simulations to predict their threshold buckling pressures and postbuckling deformations. We demonstrated that the GV diameter has an inverse cubic relation to the threshold buckling pressure, whereas length has no substantial effect. To complement these simulations, we experimentally probed the effect of geometry on the mechanical properties of GVs and the corresponding nonlinear ultrasound signals. The results of these experiments corroborate our computational predictions. This study provides fundamental insights into how geometry affects the sonomechanical properties of GVs, which, in turn, can inform further engineering of these nanostructures for high-contrast, nonlinear ultrasound imaging.

Ultrafast amplitude modulation for molecular and hemodynamic ultrasound imaging.

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 3.2 kHz. 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 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.

Self-assembly of protein superstructures by physical interactions under cytoplasm-like conditions.

The structure-driven assembly of multimeric protein complexes and the formation of intracellular phase-like protein condensates have been the subject of intense research. However, the assembly of larger superstructures comprising cellular components, such as protein nanoparticles driven by general physical rather than specific biochemical interactions, remains relatively uncharacterized. Here, we use gas vesicles (GVs)-genetically encoded protein nanoparticles that form ordered intracellular clusters-as a model system to study the forces driving multiparticle assembly under cytoplasm-like conditions. Our calculations and experimental results show that the ordered assembly of GVs can be achieved by screening their mutual electrostatic repulsion with electrolytes and creating a crowding force with dissolved macromolecules. The precise balance of these forces results in different packing configurations. Biomacromolecules such as polylysine and DNA are capable of driving GV clustering. These results provide basic insights into how physically driven interactions affect the formation of protein superstructures, offer guidance for manipulating nanoparticle assembly in cellular environments through synthetic biology methods, and inform research on the biotechnology applications of GVs.

Ultrasound Technologies for Imaging and Modulating Neural Activity.

Visualizing and perturbing neural activity on a brain-wide scale in model animals and humans is a major goal of neuroscience technology development. Established electrical and optical techniques typically break down at this scale due to inherent physical limitations. In contrast, ultrasound readily permeates the brain, and in some cases the skull, and interacts with tissue with a fundamental resolution on the order of 100 µm and 1 ms. This basic ability has motivated major efforts to harness ultrasound as a modality for large-scale brain imaging and modulation. These efforts have resulted in already-useful neuroscience tools, including high-resolution hemodynamic functional imaging, focused ultrasound neuromodulation, and local drug delivery. Furthermore, recent breakthroughs promise to connect ultrasound to neurons at the genetic level for biomolecular imaging and sonogenetic control. In this article, we review the state of the art and ongoing developments in ultrasonic neurotechnology, building from fundamental principles to current utility, open questions, and future potential.

Acoustic biosensors for ultrasound imaging of enzyme activity.

Visualizing biomolecular and cellular processes inside intact living organisms is a major goal of chemical biology. However, existing molecular biosensors, based primarily on fluorescent emission, have limited utility in this context due to the scattering of light by tissue. In contrast, ultrasound can easily image deep tissue with high spatiotemporal resolution, but lacks the biosensors needed to connect its contrast to the activity of specific biomolecules such as enzymes. To overcome this limitation, we introduce the first genetically encodable acoustic biosensors-molecules that 'light up' in ultrasound imaging in response to protease activity. These biosensors are based on a unique class of air-filled protein nanostructures called gas vesicles, which we engineered to produce nonlinear ultrasound signals in response to the activity of three different protease enzymes. We demonstrate the ability of these biosensors to be imaged in vitro, inside engineered probiotic bacteria, and in vivo in the mouse gastrointestinal tract.

Publisher Correction: Acoustic biosensors for ultrasound imaging of enzyme activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An Online Impedance Analysis and Matching System for Ultrasonic Transducers.

Electrical impedance matching of ultrasonic transducers is important in optimizing the energy consumption as well as guaranteeing equipment safety for ultrasonic systems in both laboratories and industries. The existing solutions usually rely on expensive instruments to conduct off-line impedance measurements and deploying static impedance matching networks for each specific transducer across a target frequency band. Here, we present an initial prototype of an online impedance analysis and matching system (OIAMS). In this system, an improved voltage-current method and a phase-difference method were integrated for online impedance measurement in real time, and an L-type matching circuit with variable components helped to achieve dynamic impedance matching. A feedback protocol was embedded in a microcontroller to adjust the matching strategies based on dynamic measurements, while the whole system was controlled through a software interface developed on a LabVIEW platform. Online measurement results showed that OIAMS conducted accurate impedance measurements with a relative error within 3.90% for amplitude and 13.11% for phase across the 100% bandwidth of the tested transducers. After matching, the reflected acoustic power of a 1.00-MHz transducer was reduced by over 50%, increasing the generated sound pressure level by over 2.8 dB across its 70% bandwidth.

Experimental Investigation on Graphene Oxide/SrCl2·6H2O Modified CaCl2·6H2O and the Resulting Thermal Performances.

Although the inorganic salt hydrate phase change materials (PCMs) such as CaCl2·6H2O have promising potential for thermal energy storage in building application, the issue of supercooling has restricted their practical application. In this study, graphene oxide (GO) and SrCl2·6H2O as binary nucleation agents were used to modify CaCl2·6H2O and reduce its supercooling degree. Compared with pure CaCl2·6H2O, the incorporation of graphene oxide (GO)/SrCl2·6H2O reduced the supercooling degree to 0.3 °C significantly. In addition, the supercooling degree of modified CaCl2·6H2O after 200 thermal cycles was still much lower than that of non-modified CaCl2·6H2O. From the results of differential scanning calorimetry (DSC), the latent heat value and phase change temperature of the modified CaCl2·6H2O were 207.88 J/g and 27.6 °C, respectively. Aluminum capsules were used to encapsulate the modified PCM and placed inside the composite wallboard. The thermal performances of the composite wallboard with modified PCM were investigated using infrared thermography. Experimental results showed that the average temperature difference between the top and bottom surfaces of modified CaCl2·6H2O/wallboard composite after 1 h heating was kept around 15.8 °C, while it was 4.9 °C for the control wallboard. The above test results proved that the modified CaCl2·6H2O demonstrated good thermal performance and can be used in buildings to maintain thermal comfort.