Molecular Acoustic Angiography: A New Technique for High-resolution Superharmonic Ultrasound Molecular Imaging

Ultrasound molecular imaging utilizes targeted microbubbles to bind to vascular targets such as integrins, selectins, and other extracellular binding domains. After binding, these microbubbles are typically imaged using low pressures and multi-pulse imaging sequences. In this article, we present an alternative approach for molecular imaging using ultrasound which relies on superharmonic signals produced by microbubble contrast agents. Bound bubbles were insonified near resonance using a low frequency (4 MHz) and superharmonic echoes were received at high frequencies (25–30 MHz). While this approach was observed to produce declining image intensity during repeated imaging in both in vitro and in vivo experiments due to bubble destruction, the feasibility of superharmonic molecular imaging was demonstrated for transmit pressures which are sufficiently high to induce shell disruption in bound microbubbles. This approach was validated using microbubbles targeted to the αvβ3 integrin in a rat fibrosarcoma model (n=5), and combined with superharmonic images of free microbubbles to produce high contrast, high resolution 3D volumes of both microvascular anatomy and molecular targeting. Image intensity over repeated scans and the effect of microbubble diameter were also assessed in vivo, indicating that larger microbubbles yield increased persistence in image intensity. Using ultrasound-based acoustic angiography images rather than conventional B-mode ultrasound to provide the underlying anatomical information facilitates anatomical localization of molecular markers. Quantitative analysis of relationships between microvasculature and targeting information indicated that most targeting occurred within 50 µm of a resolvable vessel (>100 µm diameter). The combined information provided by these scans may present new opportunities for analyzing relationships between microvascular anatomy and vascular targets, subject only to limitations of the current mechanically-scanned system and microbubble persistence to repeated imaging at moderate mechanical indices

Acoustic Behavior of Halobacterium salinarum Gas Vesicles in the High-Frequency Range: Experiments and Modeling

Gas vesicles (GVs) are a new and unique class of biologically derived ultrasound contrast agents with sub-micron size whose acoustic properties have not been fully elucidated. In this study, we investigated the acoustic collapse pressure and behavior of Halobacterium salinarum gas vesicles at transmit center frequencies ranging from 12.5 to 27.5 MHz. The acoustic collapse pressure was found to be above 550 kPa at all frequencies, nine-fold higher than the critical pressure observed under hydrostatic conditions. We illustrate that gas vesicles behave non-linearly when exposed to ultrasound at incident pressure ranging from 160 kPa to the collapse pressure and generate second harmonic amplitudes of −2 to −6 dB below the fundamental in media with viscosities ranging from 0.89 to 8 mPa·s. Simulations performed using a Rayleigh–Plesset-type model accounting for buckling and a dynamic finite-element analysis suggest that buckling is the mechanism behind the generation of harmonics. We found good agreement between the level of second harmonic relative to the fundamental measured at 20 MHz and the Rayleigh–Plesset model predictions. Finite-element simulations extended these findings to a non-spherical geometry, confirmed that the acoustic buckling pressure corresponds to the critical pressure under hydrostatic conditions and support the hypothesis of limited gas flow across the GV shell during the compression phase in the frequency range investigated. From simulations, estimates of GV bandwidth-limited scattering indicate that a single GV has a scattering cross section comparable to that of a red blood cell. These findings will inform the development of GV-based contrast agents and pulse sequences to optimize their detection with ultrasound

Acoustic Angiography: A New Imaging Modality for Assessing Microvasculature Architecture

The purpose of this paper is to provide the biomedical imaging community with details of a new high resolution contrast imaging approach referred to as “acoustic angiography.” Through the use of dual-frequency ultrasound transducer technology, images acquired with this approach possess both high resolution and a high contrast-to-tissue ratio, which enables the visualization of microvascular architecture without significant contribution from background tissues. Additionally, volumetric vessel-tissue integration can be visualized by using b-mode overlays acquired with the same probe. We present a brief technical overview of how the images are acquired, followed by several examples of images of both healthy and diseased tissue volumes. 3D images from alternate modalities often used in preclinical imaging, contrast-enhanced micro-CT and photoacoustics, are also included to provide a perspective on how acoustic angiography has qualitatively similar capabilities to these other techniques. These preliminary images provide visually compelling evidence to suggest that acoustic angiography may serve as a powerful new tool in preclinical and future clinical imaging

High-resolution, high-contrast ultrasound imaging using a prototype dual-frequency transducer: In vitro and in vivo studies

With recent advances in animal models of disease, there has been great interest in capabilities for high-resolution contrast-enhanced ultrasound imaging. Microbubble contrast agents are unique in that they scatter broadband ultrasound energy because of their nonlinear behavior. For optimal response, it is desirable to excite the microbubbles near their resonant frequency. To date, this has been challenging with high-frequency imaging systems because most contrast agents are resonant at frequencies in the order of several megahertz. Our team has developed a unique dual-frequency confocal transducer which enables low-frequency excitation of bubbles near their resonance with one element, and detection of their emitted high-frequency content with the second element. Using this imaging approach, we have attained an average 12.3 dB improvement in contrast-to-tissue ratios over fundamental mode imaging, with spatial resolution near that of the high-frequency element. Because this detection method does not rely on signal decorrelation, it is not susceptible to corruption by tissue motion. This probe demonstrates contrast imaging capability with significant tissue suppression, enabling high-resolution contrast-enhanced images of microvascular blood flow. Additionally, this probe can readily produce radiation force on flowing contrast agents, which may be beneficial for targeted imaging or therapy

Quantification of Microvascular Tortuosity during Tumor Evolution Using Acoustic Angiography

The recent design of ultra-broadband, multi-frequency ultrasound transducers has enabled high sensitivity, high-resolution contrast imaging, with very efficient suppression of tissue background using a technique called acoustic angiography. Here we perform the first application of acoustic angiography to evolving tumors in mice predisposed to develop mammary carcinoma, with the intent of visualizing and quantifying angiogenesis progression associated with tumor growth. Metrics compared include vascular density and two measures of vessel tortuosity quantified from segmentations of vessels traversing and surrounding 24 tumors and abdominal vessels from control mice. Quantitative morphological analysis of tumor vessels demonstrated significantly increased vascular tortuosity abnormalities associated with tumor growth with the distance metric elevated approximately 14% and the sum of angles metric increased 60% in tumor vessels versus controls. Future applications of this imaging approach may provide clinicians a new tool in tumor detection, differentiation, or evaluation, though with limited depth of penetration using the current configuration

Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI

Gas vesicles (GVs) are a unique class of gas-filled protein nanostructures that are detectable at subnanomolar concentrations and whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and MRI. Here we provide a protocol for isolating GVs from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and MRI. GVs can be isolated from natural cyanobacterial and haloarchaeal host organisms or from Escherichia coli expressing a heterologous GV gene cluster and purified using buoyancy-assisted techniques. They can then be modified by replacing surface-bound proteins with engineered, heterologously expressed variants or through chemical conjugation, resulting in altered mechanical, surface and targeting properties. Pressurized absorbance spectroscopy is used to characterize their mechanical properties, whereas dynamic light scattering (DLS)and transmission electron microscopy (TEM) are used to determine nanoparticle size and morphology, respectively. GVs can then be imaged with ultrasound in vitro and in vivo using pulse sequences optimized for their detection versus background. They can also be imaged with hyperpolarized xenon MRI using chemical exchange saturation transfer between GV-bound and dissolved xenon—a technique currently implemented in vitro. Taking 3–8 d to prepare, these genetically encodable nanostructures enable multimodal, noninvasive biological imaging with high sensitivity and potential for molecular targeting

In vivo Biodistribution of Radiolabeled Acoustic Protein Nanostructures

Purpose: Contrast-enhanced ultrasound plays an expanding role in oncology, but its applicability to molecular imaging is hindered by a lack of nanoscale contrast agents that can reach targets outside the vasculature. Gas vesicles (GVs)—a unique class of gas-filled protein nanostructures—have recently been introduced as a promising new class of ultrasound contrast agents that can potentially access the extravascular space and be modified for molecular targeting. The purpose of the present study is to determine the quantitative biodistribution of GVs, which is critical for their development as imaging agents. Procedures: We use a novel bioorthogonal radiolabeling strategy to prepare technetium-99m-radiolabeled ([99mTc])GVs in high radiochemical purity. We use single photon emission computed tomography (SPECT) and tissue counting to quantitatively assess GV biodistribution in mice. Results: Twenty minutes following administration to mice, the SPECT biodistribution shows that 84 % of [99mTc]GVs are taken up by the reticuloendothelial system (RES) and 13 % are found in the gall bladder and duodenum. Quantitative tissue counting shows that the uptake (mean ± SEM % of injected dose/organ) is 0.6 ± 0.2 for the gall bladder, 46.2 ± 3.1 for the liver, 1.91 ± 0.16 for the lungs, and 1.3 ± 0.3 for the spleen. Fluorescence imaging confirmed the presence of GVs in RES. Conclusions: These results provide essential information for the development of GVs as targeted nanoscale imaging agents for ultrasound

In vivo Biodistribution of Radiolabeled Acoustic Protein Nanostructures

Purpose: Contrast-enhanced ultrasound plays an expanding role in oncology, but its applicability to molecular imaging is hindered by a lack of nanoscale contrast agents that can reach targets outside the vasculature. Gas vesicles (GVs)—a unique class of gas-filled protein nanostructures—have recently been introduced as a promising new class of ultrasound contrast agents that can potentially access the extravascular space and be modified for molecular targeting. The purpose of the present study is to determine the quantitative biodistribution of GVs, which is critical for their development as imaging agents. Procedures: We use a novel bioorthogonal radiolabeling strategy to prepare technetium-99m-radiolabeled ([99mTc])GVs in high radiochemical purity. We use single photon emission computed tomography (SPECT) and tissue counting to quantitatively assess GV biodistribution in mice. Results: Twenty minutes following administration to mice, the SPECT biodistribution shows that 84 % of [99mTc]GVs are taken up by the reticuloendothelial system (RES) and 13 % are found in the gall bladder and duodenum. Quantitative tissue counting shows that the uptake (mean ± SEM % of injected dose/organ) is 0.6 ± 0.2 for the gall bladder, 46.2 ± 3.1 for the liver, 1.91 ± 0.16 for the lungs, and 1.3 ± 0.3 for the spleen. Fluorescence imaging confirmed the presence of GVs in RES. Conclusions: These results provide essential information for the development of GVs as targeted nanoscale imaging agents for ultrasound

Is older age associated with COVID-19 mortality in the absence of other risk factors? General population cohort study of 470,034 participants

Introduction: Older people have been reported to be at higher risk of COVID-19 mortality. This study explored the factors mediating this association and whether older age was associated with increased mortality risk in the absence of other risk factors. Methods: In UK Biobank, a population cohort study, baseline data were linked to COVID-19 deaths. Poisson regression was used to study the association between current age and COVID-19 mortality. Results: Among eligible participants, 438 (0.09%) died of COVID-19. Current age was associated exponentially with COVID-19 mortality. Overall, participants aged ≥75 years were at 13-fold (95% CI 9.13–17.85) mortality risk compared with those <65 years. Low forced expiratory volume in 1 second, high systolic blood pressure, low handgrip strength, and multiple long-term conditions were significant mediators, and collectively explained 39.3% of their excess risk. The associations between these risk factors and COVID-19 mortality were stronger among older participants. Participants aged ≥75 without additional risk factors were at 4-fold risk (95% CI 1.57–9.96, P = 0.004) compared with all participants aged <65 years. Conclusions: Higher COVID-19 mortality among older adults was partially explained by other risk factors. ‘Healthy’ older adults were at much lower risk. Nonetheless, older age was an independent risk factor for COVID-19 mortality

The evolution of irradiance detection: melanopsin and the non-visual opsins

Circadian rhythms are endogenous 24 h cycles that persist in the absence of external time cues. These rhythms provide an internal representation of day length and optimize physiology and behaviour to the varying demands of the solar cycle. These clocks require daily adjustment to local time and the primary time cue (zeitgeber) used by most vertebrates is the daily change in the amount of environmental light (irradiance) at dawn and dusk, a process termed photoentrainment. Attempts to understand the photoreceptor mechanisms mediating non-image-forming responses to light, such as photoentrainment, have resulted in the discovery of a remarkable array of different photoreceptors and photopigment families, all of which appear to use a basic opsin/vitamin A-based photopigment biochemistry. In non-mammalian vertebrates, specialized photoreceptors are located within the pineal complex, deep brain and dermal melanophores. There is also strong evidence in fish and amphibians for the direct photic regulation of circadian clocks in multiple tissues. By contrast, mammals possess only ocular photoreceptors. However, in addition to the image-forming rods and cones of the retina, there exists a third photoreceptor system based on a subset of melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). In this review, we discuss the range of vertebrate photoreceptors and their opsin photopigments, describe the melanopsin/pRGC system in some detail and then finally consider the molecular evolution and sensory ecology of these non-image-forming photoreceptor systems