Current Development and Applications of Super-Resolution Ultrasound Imaging.

Abnormal changes of the microvasculature are reported to be key evidence of the development of several critical diseases, including cancer, progressive kidney disease, and atherosclerotic plaque. Super-resolution ultrasound imaging is an emerging technology that can identify the microvasculature noninvasively, with unprecedented spatial resolution beyond the acoustic diffraction limit. Therefore, it is a promising approach for diagnosing and monitoring the development of diseases. In this review, we introduce current super-resolution ultrasound imaging approaches and their preclinical applications on different animals and disease models. Future directions and challenges to overcome for clinical translations are also discussed.

Ultrasound super-resolution imaging provides a noninvasive assessment of renal microvasculature changes during mouse acute kidney injury.

Acute kidney injury (AKI) is a risk factor for the development of chronic kidney disease (CKD). One mechanism for this phenomenon is renal microvascular rarefaction and subsequent chronic impairment in perfusion. However, diagnostic tools to monitor the renal microvasculature in a noninvasive and quantitative manner are still lacking. Ultrasound super-resolution imaging is an emerging technology that can identify microvessels with unprecedented resolution. Here, we applied this imaging technique to identify microvessels in the unilateral ischemia-reperfusion injury mouse model of AKI-to-CKD progression in vivo. Kidneys from 21 and 42 day post- ischemia-reperfusion injury, the contralateral uninjured kidneys, and kidneys from sham-operated mice were examined by ultrasound super-resolution and histology. Renal microvessels were successfully identified by this imaging modality with a resolution down to 32 µm. Renal fibrosis was observed in all kidneys with ischemia-reperfusion injury and was associated with a significant reduction in kidney size, cortical thickness, relative blood volume, and microvascular density as assessed by this imaging. Tortuosity of the cortical microvasculature was also significantly increased at 42 days compared to sham. These vessel density measurements correlated significantly with CD31 immunohistochemistry (R2=0.77). Thus, ultrasound super-resolution imaging provides unprecedented resolution and is capable of noninvasive quantification of renal vasculature changes associated with AKI-to-CKD progression in mice. Hence, this technique could be a promising diagnostic tool for monitoring progressive kidney disease.

Multistage adaptive noise reduction technique for optical resolution photoacoustic microscopy.

Photoacoustic microscopy has received great attention due to the benefits of the optical resolution contrast as well as its superior spatial resolution and relatively deep depth. Like other imaging modalities, photoacoustic images suffer from noise, and filtering techniques are required to remove them. To overcome the noise, we proposed a combination of filters, including an adaptive median filter, an effective filter for impulsive noise, and a nonlocal means filter, an effective filter for background noise, for noise removal and image quality enhancement. Our proposed method enhanced the signal-to-noise ratio by 16 dB in an in vivo study compared to the traditional image reconstruction approach and preserved the image detail with minimal blurring, which usually occurs when filtering. These experimental results verified that the proposed adaptive multistage denoising techniques could effectively improve image quality under noisy data acquisition conditions, providing a strong foundation for photoacoustic microscopy with limited laser power.

Design of a Volumetric Imaging Sequence Using a Vantage-256 Ultrasound Research Platform Multiplexed With a 1024-Element Fully Sampled Matrix Array.

Ultrasound imaging using a matrix array allows real-time multi-planar volumetric imaging. To enhance image quality, the matrix array should provide fast volumetric ultrasound imaging with spatially consistent focusing in the lateral and elevational directions. However, because of the significantly increased data size, dealing with massive and continuous data acquisition is a significant challenge. We have designed an imaging acquisition sequence that handles volumetric data efficiently using a single 256-channel Verasonics ultrasound research platform multiplexed with a 1024-element matrix array. The developed sequence has been applied for building an ultrasonic pupilometer. Our results demonstrate the capability of the developed approach for structural visualization of an ex vivo porcine eye and the temporal response of the modeled eye pupil with moving iris at the volume rate of 30 Hz. Our study provides a fundamental ground for researchers to establish their own volumetric ultrasound imaging platform and could stimulate the development of new volumetric ultrasound approaches and applications.

Validation of Ultrasound Super-Resolution Imaging of Vasa Vasorum in Rabbit Atherosclerotic Plaques.

Acute coronary syndromes and strokes are mainly caused by atherosclerotic plaque (AP) rupture. Abnormal increase of vasa vasorum (VV) is reported as a key evidence of plaque progression and vulnerability. However, due to their tiny size, it is still challenging to noninvasively identify VV near the major vessels. Ultrasound super resolution (USR), a technique that provides high spatial resolution beyond the acoustic diffraction limit, demonstrated an adequate spatial resolution for VV detection in early studies. However, a thorough validation of this technology in the plaque model is particularly needed in order to continue further extended preclinical studies. In this letter, we present an experiment protocol that verifies the USR technology for VV identification with subsequent histology and ex vivo micro-computed tomography ( µ CT). Deconvolution-based USR imaging was applied on two rabbits to identify the VV near the AP in the femoral artery. Histology and ex vivo µ CT imaging were performed on excised femoral tissue to validate the USR technique both pathologically and morphologically. This established validation protocol could facilitate future extended preclinical studies toward the clinical translation of USR imaging for VV identification.

Super-resolution ultrasound imaging method for microvasculature in vivo with a high temporal accuracy.

Traditional ultrasound imaging techniques are limited in spatial resolution to visualize angiogenic vasa vasorum that is considered as an important marker for atherosclerotic plaque progression and vulnerability. The recently introduced super-resolution imaging technique based on microbubble center localization has shown potential to achieve unprecedented high spatial resolution beyond the acoustic diffraction limit. However, a major drawback of the current super-resolution imaging approach is low temporal resolution because it requires a large number of imaging frames. In this study, a new imaging sequence and signal processing approach for super-resolution ultrasound imaging are presented to improve temporal resolution by employing deconvolution and spatio-temporal-interframe-correlation based data acquisition. In vivo feasibility of the developed technology is demonstrated and evaluated in imaging vasa vasorum in the rabbit atherosclerosis model. The proposed method not only identifies a tiny vessel with a diameter of 41 µm, 5 times higher spatial resolution than the acoustic diffraction limit at 7.7 MHz, but also significantly improves temporal resolution that allows for imaging vessels over cardiac motion.

Review: optically-triggered phase-transition droplets for photoacoustic imaging.

Optically-triggered phase-transition droplets have been introduced as a promising contrast agent for photoacoustic and ultrasound imaging that not only provide significantly enhanced contrast but also have potential as photoacoustic theranostic molecular probes incorporated with targeting molecules and therapeutics. For further understanding the dynamics of optical droplet vaporization process, an innovative, methodical analysis by concurrent acoustical and ultrafast optical recordings, comparing with a theoretical model has been employed. In addition, the repeatability of the droplet vaporization-recondensation process, which enables continuous photoacoustic imaging has been studied through the same approach. Further understanding the underlying physics of the optical droplet vaporization and associated dynamics may guide the optimal design of the droplets. Some innovative approaches in preclinical studies have been recently demonstrated, including sono-photoacoustic imaging, dual-modality of photoacoustic and ultrasound imaging, and super-resolution photoacoustic imaging. In this review, current development of optically triggered phase-transition droplets and understanding on the vaporization dynamics, their applications are introduced and future directions are discussed.

Photostable, hydrophilic, and near infrared quaterrylene-based dyes for photoacoustic imaging.

Novel near-infrared contrast agents based on the quaterrylene structure were strategically developed and tested for high photo-stability. Both a dendrimeric quaterrylene molecule, QR-G2-COOH, and a small molecule cationic quaterrylene dye, QR-4PyC4, remain optically stable and continue to generate a competitive photoacoustic response when irradiated by short near-infrared laser pulses for a relatively long time in an in-vitro cell study, unlike indocyanine green that rapidly decreases photoacoustic signal amplitude. The small molecule dye, QR-4PyC4 exhibits not only significantly higher cellular uptake rate than QR-G2-COOH and indocyanine green, but also low toxicity at a concentration of up to 10 µM. The dendrimeric dye, QR-G2-COOH that has surface functional groups available for conjugation with targeting and therapeutic agents shows the highest photoacoustic amplitude with high optical stability. Therefore, QR-4PyC4 can be a promising universal, sensitive and reliable photoacoustic contrast agent and QR-G2-COOH has great potential as a nano-platform with stable photoacoustic imaging capability.

Recent Development of Technology and Application of Photoacoustic Molecular Imaging Toward Clinical Translation.

The deep imaging capability and optical absorption contrast offered by photoacoustic imaging promote the use of this technology in clinical applications. By exploiting the optical absorption properties of endogenous chromophores such as hemoglobin and lipid, molecular information at a depth of a few centimeters can be unveiled. This information shows promise to reveal lesions indicating early stage of various human diseases, such as cancer and atherosclerosis. In addition, the use of exogenous contrast agents can further extend the capability of photoacoustic imaging in clinical diagnosis and treatment. In this review, the current state of the art and applications of photoacoustic molecular probes will be critically reviewed, as well as some spearheading translational efforts that have taken place over the past 5 years. Some of the most critical barriers to clinical translation of this novel technology will be discussed, and some thoughts will be given on future endeavors and pathways.

A Light Illumination Enhancement Device for Photoacoustic Imaging: In Vivo Animal Study.

Photoacoustic (PA) imaging detects acoustic signals generated by thermal expansion of a light-excited tissue or contrast agents. PA signal amplitude and image quality directly depend on the light fluence at the target depth. With conventional PA imaging systems, approximately 30% energy of incident light at the near-infrared region would be lost due to reflection on the skin surface. Such light loss directly leads to a reduction of PA signal and image quality. A new light delivery scheme that collects and redistributes reflected light energy was recently suggested, which is called the light catcher. In our previous study, proof of concept using a finite-element simulation model was shown and a laboratory-built prototype of the light catcher was applied on tissue-mimicking phantoms. In this paper, we present an elaborate prototype of a high-frequency PA probe with the light catcher fabricated using 3-D printing technology, which is conformal to a subcutaneous tumor in mice. The in vivo usefulness of the developed prototype was evaluated in a mouse tumor model. Equipped with the light catcher, PA signal amplitude from the clinical photosensitizer injected into the mouse tumor was enhanced by 33.7%, which is approximately equivalent to the percent light loss due to reflection on the skin.

Handheld photoacoustic tomography probe built using optical-fiber parallel acoustic delay lines.

The development of the first miniaturized parallel acoustic delay line (PADL) probe for handheld photoacoustic tomography (PAT) is reported. Using fused-silica optical fibers with low acoustic attenuation, we constructed two arrays of eight PADLs. Precision laser micromachining was conducted to produce robust and accurate mechanical support and alignment structures for the PADLs, with minimal acoustic distortion and interchannel coupling. The 16 optical-fiber PADLs, each with a different time delay, were arranged to form one input port and two output ports. A handheld PADL probe was constructed using two single-element transducers and two data acquisition channels (equal to a channel reduction ratio of 8∶1). Photoacoustic (PA) images of a black-ink target embedded in an optically scattering phantom were successfully acquired. After traveling through the PADLs, the eight channels of differently time-delayed PA signals reached each single-element ultrasonic transducer in a designated nonoverlapping time series, allowing clear signal separation for PA image reconstruction. Our results show that the PADL technique and the handheld probe can potentially enable real-time PAT, while significantly reducing the complexity and cost of the ultrasound receiver system.

Lung macrophages "digest" carbon nanotubes using a superoxide/peroxynitrite oxidative pathway.

In contrast to short-lived neutrophils, macrophages display persistent presence in the lung of animals after pulmonary exposure to carbon nanotubes. While effective in the clearance of bacterial pathogens and injured host cells, the ability of macrophages to "digest" carbonaceous nanoparticles has not been documented. Here, we used chemical, biochemical, and cell and animal models and demonstrated oxidative biodegradation of oxidatively functionalized single-walled carbon nanotubes via superoxide/NO* → peroxynitrite-driven oxidative pathways of activated macrophages facilitating clearance of nanoparticles from the lung.

Narrow size distribution of microbubbles for enhancement of harmonic imaging.

An ultrasound microbubble contrast agent is a promising technique in clinical diagnosis because ultrasound in combination with microbubbles enhances the ultrasound backscatter to produce an increased contrast images. In this study, we developed phospholipid-based microbubbles showing a relatively narrow size distribution of 0.8-1.3 microm. The optimal resonance frequency of developed microbubbles was determined to be 2.5-3.0 MHz by measuring echo signals at various frequencies. Ultrasound harmonic imaging was performed in a vessel phantom at the optimal resonance frequencies. Microbubble contrast-enhanced ultrasound images visualized a vessel tube clearly and demonstrated much improved image quality, compared to the control. In conclusion, the ultrasonography in the harmonic mode is capable of maximally resonating micrbubbles with a narrow size distribution at a specific frequency for enhanced ultrasound imaging.