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.

Laser Tracker and Terrestrial Laser Scanner Range Error Evaluation by Stitching.

Laser trackers (LTs) are dimensional measurement instruments commonly employed in the manufacture and assembly of large structures. Terrestrial laser scanners (TLSs) are a related class of dimensional measurement instruments more commonly employed in surveying, reverse engineering, and forensics. Commercially available LTs typically have measurement ranges of up to 80 m. The measurement ranges of TLSs vary from about 50 m to several hundred meters, with some extending as far as several kilometers. It is difficult, if not impossible, to construct long reference lengths to evaluate the ranging performances of these instruments over that distance. In this context, we explore the use of stitching errors (i.e., stacking errors in adjoining or overlapping short lengths) and stitching lengths (i.e., constructing long reference lengths from multiple positions of a reference instrument by registration) to evaluate these instruments. Through experimental data and a discussion on uncertainty, we show that stitching is indeed a viable option to evaluate the ranging performances of LTs and TLSs.

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.

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.

Utilization of the Lempert Maneuver for Benign Paroxysmal Positional Vertigo in the Emergency Department.

Benign paroxysmal positional vertigo (BPPV) is a common cause of vertigo. Symptoms from BPPV lead to Emergency Department (ED) visits, and persistence of symptoms, particularly in the elderly, may impact patient disposition. We describe the techniques used in the case of a 72-year-old male with dizziness, who had symptom resolution, and was able to be safely discharged after a Lempert maneuver (barbeque (BBQ) roll) was performed in the ED setting. The patient presented to the ED with left gaze nystagmus, and otherwise normal evaluation results. Physical therapy was consulted, and their initial evaluation indicated right horizontal canalithiasis noted by fatiguing right, geotropic nystagmus, but the patient was unable to tolerate further testing due to vomiting. Antiemetic medications were administered and at his follow-up examination an hour later, a total of three Lempert maneuvers were performed, resulting in total symptom resolution. Successful utilization of the Lempert maneuver to treat BPPV can help to reduce ED length of stay and increase patient satisfaction. Because of this, the Lempert maneuver should be considered a fast, cost-effective, and safe method of alleviating BPPV symptoms.

Forgotten books: The application of unseen species models to the survival of culture.

The study of ancient cultures is hindered by the incomplete survival of material artifacts, so we commonly underestimate the diversity of cultural production in historic societies. To correct this survivorship bias, we applied unseen species models from ecology to gauge the loss of narratives from medieval Europe, such as the romances about King Arthur. The estimates obtained are compatible with the scant historic evidence. In addition to events such as library fires, we identified the original evenness of cultural populations as an overlooked factor in these assemblages' stability in the face of immaterial loss. We link the elevated evenness in island literatures to analogous accounts of ecological and cultural diversity in insular communities. These analyses call for a wider application of these methods across the heritage sciences.

Ultrasensitive ultrasound imaging of gene expression with signal unmixing.

Acoustic reporter genes (ARGs) that encode air-filled gas vesicles enable ultrasound-based imaging of gene expression in genetically modified bacteria and mammalian cells, facilitating the study of cellular function in deep tissues. Despite the promise of this technology for biological research and potential clinical applications, the sensitivity with which ARG-expressing cells can be visualized is currently limited. Here we present burst ultrasound reconstructed with signal templates (BURST)-an ARG imaging paradigm that improves the cellular detection limit by more than 1,000-fold compared to conventional methods. BURST takes advantage of the unique temporal signal pattern produced by gas vesicles as they collapse under acoustic pressure above a threshold defined by the ARG. By extracting the unique pattern of this signal from total scattering, BURST boosts the sensitivity of ultrasound to image ARG-expressing cells, as demonstrated in vitro and in vivo in the mouse gastrointestinal tract and liver. Furthermore, in dilute cell suspensions, BURST imaging enables the detection of gene expression in individual bacteria and mammalian cells. The resulting abilities of BURST expand the potential use of ultrasound for non-invasive imaging of cellular functions.

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.

Terrestrial laser scanner calibration and performance evaluation using the network method.

In this paper, we discuss two aspects concerning terrestrial laser scanners (TLSs) - error model calibration and performance evaluation. Error model calibration is the process of determining parameters of an error model to improve the accuracy of TLSs. Performance evaluation refers to a series of tests to determine if a TLS meets specifications provided by the manufacturer. Both procedures can be accomplished using a network of stationary targets whose locations are known from a prior calibration using another method/instrument. This paper explores the question of whether the network (i.e., target locations) must be calibrated using an instrument of higher accuracy such as a laser tracker (LT) or whether the TLS under study is itself suitable for network calibration. Regardless of whether an LT or a TLS is used, the calibration is performed from target measurements made from multiple locations of the instrument to average out systematic errors and reduce the uncertainties in target coordinates. Such multi-position measurements on stationary targets is referred to as the network method. We provide guidance on when the TLS is sufficient for network calibration and when an LT may be necessary for performance evaluation purposes.

Improvised Long Test Lengths via Stitching Scale Bar Method: Performance Evaluation of Terrestrial Laser Scanners per ASTM E3125-17.

Periodic performance evaluation is a critical issue for ensuring the reliability of data from terrestrial laser scanners (TLSs). With the recent introduction of the ASTM E3125-17 standard, there now exist standardized test procedures for this purpose. Point-to-point length measurement is one test method described in that documentary standard. This test is typically performed using a long scale bar (typically 2 m or longer) with spherical targets mounted on both ends. Long scale bars can become unwieldy and vary in length due to gravity loading, fixture forces, and environmental changes. In this paper, we propose a stitching scale bar (SSB) method in which a short scale bar (approximately 1 m or smaller) can provide a spatial length reference several times its length. The clear advantages of a short scale bar are that it can be calibrated in a laboratory and has potential long-term stability. An essential requirement when stitching a short scale bar is that the systematic errors in TLSs do not change significantly over short distances. We describe this requirement in this paper from both theoretical and experimental perspectives. Based on this SSB method, we evaluate the performance of a TLS according to the ASTM E3125-17 standard by stitching a 1.15 m scale bar to form a 2.3 m reference length. For comparison, a single 2.3 m scale bar is also employed for direct measurements without stitching. Experimental results show a maximum deviation of 0.072 mm in length errors between the two approaches, which is an order of magnitude smaller than typical accuracy specifications for TLSs.

Ultrasound imaging of gene expression in mammalian cells.

The study of cellular processes occurring inside intact organisms requires methods to visualize cellular functions such as gene expression in deep tissues. Ultrasound is a widely used biomedical technology enabling noninvasive imaging with high spatial and temporal resolution. However, no genetically encoded molecular reporters are available to connect ultrasound contrast to gene expression in mammalian cells. To address this limitation, we introduce mammalian acoustic reporter genes. Starting with a gene cluster derived from bacteria, we engineered a eukaryotic genetic program whose introduction into mammalian cells results in the expression of intracellular air-filled protein nanostructures called gas vesicles, which produce ultrasound contrast. Mammalian acoustic reporter genes allow cells to be visualized at volumetric densities below 0.5% and permit high-resolution imaging of gene expression in living animals.

METROLOGICAL EVALUATION OF CONTRAST TARGET CENTER ALGORITHM FOR TERRESTRIAL LASER SCANNERS.

Terrestrial Laser Scanners (TLSs) are used in a variety of large scale scanning applications such as reverse engineering, assembly of aircraft or ships and surveying. Contrast targets are used with such instruments for enabling scene registration or to establish a scale when used on a scale bar. Currently, the algorithms to calculate the centers of contrast targets (CCT) are either proprietary to the original equipment manufacturers (OEMs) or not precise and accurate. Some of these algorithms may also operate only on OEM's proprietary data format. To overcome these limitations, a novel algorithm was developed at the National Institute of Standards and Technology (NIST) to calculate the center of contrast targets. Several targets in various orientations were scanned by one TLS and their centers were calculated by both the NIST algorithm and one OEM software. The results show that the NIST algorithm is robust, addresses many data quality issues and has better precision than the OEM software in most cases.

Nonlinear X-wave ultrasound imaging of acoustic biomolecules.

The basic physics of sound waves enables ultrasound to visualize biological tissues with high spatial and temporal resolution. Recently, this capability was enhanced with the development of acoustic biomolecules - proteins with physical properties enabling them to scatter sound. 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. Acoustical methods for the in vivo detection of GVs are now required to maximize the impact of this technology in biology and medicine. 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. 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. 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. 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. 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. We show in both simulations and experiments that residual xAM nonlinearity due to wave propagation decreases as the plane-wave cross-propagation angle increases. 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. 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. These results advance the physical facet of the emerging field of biomolecular ultrasound, and are also relevant to synthetic ultrasound contrast agents.

Concept to Commercialization of an Artifact for Evaluating Three-Dimensional Imaging Systems per ASTM E3125-17.

The relative-range error test is one of several tests described in the ASTM E3125-2017 standard for performance evaluation of spherical coordinate three-dimensional (3D) imaging systems such as terrestrial laser scanners (TLS). We designed a new artifact, called the plate-sphere target, that allows the realization of the relative-range error tests quickly and efficiently without the need for alignment at each position of the test.Use of a simple planar/plate target requires careful alignment of the target at each position of the relative-range error test, which is labor-intensive and time-consuming. This new artifact significantly reduces the time required to perform the test, from a matter of about 2 h to less than 30 min while resulting in similar test uncertainty values.The plate-sphere target was conceived and initially developed at the National Institute of Standards and Technology (NIST), improved based on feedback from collaborators at the National Research Council (NRC) of Canada and TLS manufacturers, and commercialized by Bal-tec Inc.This new artifact will save users and manufacturers of TLSs considerable time and money.

A very large-scale microelectrode array for cellular-resolution electrophysiology.

In traditional electrophysiology, spatially inefficient electronics and the need for tissue-to-electrode proximity defy non-invasive interfaces at scales of more than a thousand low noise, simultaneously recording channels. Using compressed sensing concepts and silicon complementary metal-oxide-semiconductors (CMOS), we demonstrate a platform with 65,536 simultaneously recording and stimulating electrodes in which the per-electrode electronics consume an area of 25.5 µm by 25.5 µm. Application of this platform to mouse retinal studies is achieved with a high-performance processing pipeline with a 1 GB/s data rate. The platform records from 65,536 electrodes concurrently with a ~10 µV r.m.s. noise; senses spikes from more than 34,000 electrodes when recording across the entire retina; automatically sorts and classifies greater than 1700 neurons following visual stimulation; and stimulates individual neurons using any number of the 65,536 electrodes while observing spikes over the entire retina. The approaches developed here are applicable to other electrophysiological systems and electrode configurations.

Determining geometric error model parameters of a terrestrial laser scanner through Two-face, Length-consistency, and Network methods.

Terrestrial laser scanners (TLS) are increasingly used in large-scale manufacturing and assembly where required measurement uncertainties are on the order of few tenths of a millimeter or smaller. In order to meet these stringent requirements, systematic errors within a TLS are compensated in-situ through self-calibration. In the Network method of self-calibration, numerous targets distributed in the work-volume are measured from multiple locations with the TLS to determine parameters of the TLS error model. In this paper, we propose two new self-calibration methods, the Two-face method and the Length-consistency method. The Length-consistency method is proposed as a more efficient way of realizing the Network method where the length between any pair of targets from multiple TLS positions are compared to determine TLS model parameters. The Two-face method is a two-step process. In the first step, many model parameters are determined directly from the difference between front-face and back-face measurements of targets distributed in the work volume. In the second step, all remaining model parameters are determined through the Length-consistency method. We compare the Two-face method, the Length-consistency method, and the Network method in terms of the uncertainties in the model parameters, and demonstrate the validity of our techniques using a calibrated scale bar and front-face back-face target measurements. The clear advantage of these self-calibration methods is that a reference instrument or calibrated artifacts are not required, thus significantly lowering the cost involved in the calibration process.

Relative range error evaluation of terrestrial laser scanners using a plate, a sphere, and a novel dual-sphere-plate target.

Terrestrial laser scanners (TLS) are a class of 3D imaging systems that produce a 3D point cloud by measuring the range and two angles to a point. The fundamental measurement of a TLS is range. Relative range error is one component of the overall range error of TLS and its estimation is therefore an important aspect in establishing metrological traceability of measurements performed using these systems. Target geometry is an important aspect to consider when realizing the relative range tests. The recently published ASTM E2938-15 mandates the use of a plate target for the relative range tests. While a plate target may reasonably be expected to produce distortion free data even at far distances, the target itself needs careful alignment at each of the relative range test positions. In this paper, we discuss relative range experiments performed using a plate target and then address the advantages and limitations of using a sphere target. We then present a novel dual-sphere-plate target that draws from the advantages of the sphere and the plate without the associated limitations. The spheres in the dual-sphere-plate target are used simply as fiducials to identify a point on the surface of the plate that is common to both the scanner and the reference instrument, thus overcoming the need to carefully align the target.