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espanolEste articulo presentan algunos de los aspectos mas sobresalientes de la investigacion y desarrollo de la acustica subacuatica, asi como reconoce el merito de miembros de la Sociedad Americana de Acustica (Acoustics Society of America, ASA), quienes contribuyeron con su trabajo durante el periodo de la Segunda Guerra Mundial. Los resultados de tal trabajo se convirtieron en logros cientificos y de ingenieria que aparecen en literatura sobre acustica. EnglishThis paper presents some of the highlights about research and development of sub-aquatic acoustics, as well as it gives recognition to the members of the Acoustical Society of America (ASA), who contributed with their work along the Second World War. The results of such work have turned into scientific and engineering achievements, which are published in literature about acoustics.

Methods to noninvasively increase and assess T cell infiltration are critical for success of adoptive cell therapy (ACT) of solid tumors where only a small fraction of adoptive T cells typically accumulates at the target. We developed an approach based on photoacoustic (PA) and ultrasound (US) imaging to visualize magnetic field driven accumulation of T cells tagged with photo-magnetic nanoparticles (PMNPs). Specifically, Au@Fe3O4 shell-core PMNPs (200 nm diameter), absorbing at 1064 nm wavelength, were synthesized and characterized using a UV-Vis-NIR spectrophotometry, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Ovalbumin (OVA)-targeted PMNP-tagged OT1 murine primary T cells were injected intravenously into OVA-expressing tumor-bearing C57BL/6 mice. US/PA imaging (20 MHz, 1064 nm and 680–970 nm, Vevo LAZR, Visualsonics Inc.) of the primary tumor and regional lymph nodes showed accumulation of PMNP-tagged T cells. Our results indicate feasibility of the T cell tagging with PMNPs and magnetic delivery of adoptive PMNP-tagged T cells using US/PA imaging thus providing critical imaging feedback to improve the adoptive T cell therapy of solid tumors. Overall, our studies show that a synergistic combination of US/PA imaging and magnetic delivery of nanoparticle (NP)-tagged adoptive T cells can expedite development, translation, and expansion of ACT.

Photoacoustic (PA) imaging is a functional and molecular imaging technique capable of high sensitivity and spatiotemporal resolution at depth. Widespread use of PA imaging, however, is limited by currently available contrast agents, which either lack PA-signal-generation ability for deep imaging or their absorbance spectra overlap with hemoglobin, reducing sensitivity. Here we report on a PA contrast agent based on targeted liposomes loaded with J-aggregated indocyanine green (ICG) dye (i.e., PAtrace) that we synthesized, bioconjugated, and characterized to addresses these limitations. We then validated PAtrace in phantom, in vitro, and in vivo PA imaging environments for both spectral unmixing accuracy and targeting efficacy in a folate receptor alpha-positive ovarian cancer model. These study results show that PAtrace concurrently provides significantly improved contrast-agent quantification/sensitivity and SO2 estimation accuracy compared to monomeric ICG. PAtrace’s performance attributes and composition of FDA-approved components make it a promising agent for future clinical molecular PA imaging., Photoacoustic imaging is limited by a lack of contrast agents which can enable combined molecular and physiological imaging at depth. Here the authors address these limitations by developing and validating a contrast agent based on targeted liposomes loaded with J-aggregated indocyanine green dye.

Photoacoustic visualization of nanoparticles is capable of high contrast imaging at depth greater than that of traditional optical imaging techniques. Identifying the impact of various parameters on the photoacoustic signal is crucial in the design of effective medical imaging and diagnostics. Here, we develop a complete model of Fourier heat conduction incorporating the interfacial thermal resistance and photoacoustic equation for core-shell nanospheres in a fluid under nanosecond pulsed laser illumination. An analytical solution is obtained, elucidating the contribution of each region (core, shell, or the fluid) in the generation of the photoacoustic signal. The model reveals that the sharper the laser pulse temporal waveform is, the higher the sensitivity of the generated photoacoustic signal will be to the interfacial thermal resistance, and, thus, the higher the possibility of photoacoustic signal amplification will be using silica-coating. The comprehensive model and adopted analytical solution reveal the underlying physics of the photoacoustic signal generation form core-shell nanosphere systems. Photoacoustic imaging of colloidal nanosystems is a useful tool for biological applications, yet current models of the photo-induced thermal processes contain un-physical assumptions. Here, the authors propose a model capable of disentangling the role of the nanoparticle, shell, surrounding material, and laser pulse properties.

This paper introduces a configurable combined laser, ultrasound, and elasticity (CLUE) imaging platform. The CLUE platform enables imaging sequences capable of simultaneously providing quantitative acoustic, optical, and mechanical contrast for comprehensive diagnosis and monitoring of complex diseases, such as cancer. The CLUE imaging platform was developed on a Verasonics ultrasound scanner integrated with a pulsed laser, and it was designed to be modular and scalable to allow researchers to create their own specific imaging sequences efficiently. The CLUE imaging platform and sequence were demonstrated in a tissue-mimicking phantom containing a stiff inclusion labeled with optically-activated nanodroplets and in an ex vivo mouse spleen. We have shown that CLUE imaging can simultaneously capture multi-functional imaging signals providing quantitative information on tissue.

Perfluorocarbon nanodroplets (PFCnDs) are phase-change contrast agents that have the potential to enable extravascular contrast-enhanced ultrasound and photoacoustic (US/PA) imaging. Producing consistently small, monodisperse PFCnDs remains a challenge without resorting to technically challenging methods. We investigated the impact of variable shell composition on PFCnD size and US/PA image properties. Our results suggest that increasing the molar percentage of PEGylated lipid reduces the size and size variance of PFCnDs. Furthermore, our imaging studies revealed that nanodroplets with more PEGylated lipids produce increased US/PA signal compared with those with the standard formulation. Finally, we highlight the ability of this approach to facilitate US/PA imaging in a murine model of breast cancer. These data indicate that, through a facile synthesis process, it is possible to produce monodisperse, small-sized PFCnDs. Novel in their simplicity, these methods may promote the use of PFCnDs among a broader user base to study a variety of extravascular phenomena.

Glaucoma is the second leading cause of blindness in the world. Disease progression is associated with reduced cellularity in the trabecular meshwork (TM), a fluid drainage tissue in the anterior eye. A promising therapy seeks to deliver stem cells to the TM to regenerate the tissue and restore its function. However, like many stem cell-based regenerative therapies, preclinical development relies heavily on histology to evaluate outcomes. To expedite clinical translation, we are developing an ultrasound/photoacoustic (US/PA) imaging platform for longitudinal tracking of stem cells in the anterior eye. Methods: Mesenchymal stem cells (MSCs) were labeled with gold nanospheres in vitro and injected through the cornea into the anterior chamber of ex vivo porcine eyes. Physiological pressure was imposed to mimic in vivo conditions. AuNS-labeled MSCs were injected through the cornea while single-wavelength US/PA images were acquired. At 5 hours post-injection, three-dimensional multi-wavelength US/PA datasets were acquired and spectroscopic analysis was used to detect AuNS-labeled MSCs. US/PA results were compared to fluorescent microscopy. Results: The US/PA imaging platform was able to provide real-time monitoring of the stem cell injection and distinguish AuNS-labeled MSCs from highly absorbing background tissues in the anterior segment. Conclusion: Our US/PA imaging approach can inform preclinical studies of stem cell therapies for glaucoma treatment, motivating further development of this theranostic imaging tool for ophthalmic applications.

Phase-change perfluorohexane nanodroplets (PFHnDs) are a new class of recondensable submicrometer-sized contrast agents that have potential for contrast-enhanced and super-resolution ultrasound imaging with an ability to reach extravascular targets. The PFHnDs can be optically triggered to undergo vaporization, resulting in spatially stationary, temporally transient microbubbles. The vaporized PFHnDs are hyperechoic in ultrasound imaging for several to hundreds of milliseconds before recondensing to their native, hypoechoic, liquid nanodroplet state. The decay of echogenicity, i.e., the dynamic behavior of the ultrasound signal from optically triggered PFHnDs in ultrasound imaging, can be captured using high-frame-rate ultrasound imaging. We explore the possibility to manipulate the echogenicity dynamics of optically triggered PFHnDs in ultrasound imaging by changing the phase of the ultrasound imaging pulse. Specifically, the ultrasound imaging system was programmed to transmit two imaging pulses with inverse polarities. We show that the imaging pulse phase can affect the amplitude and the temporal behavior of PFHnD echogenicity in ultrasound imaging. The results of this study demonstrate that the ultrasound echogenicity is significantly increased (about 78% improvement) and the hyperechoic timespan of optically triggered PFHnDs is significantly longer (about four times) if the nanodroplets are imaged by an ultrasound pulse starting with rarefactional pressure versus a pulse starting with compressional pressure. Our finding has direct and significant implications for contrast-enhanced ultrasound imaging of droplets in applications such as super-resolution imaging and molecular imaging where detection of individual or low-concentration PFHnDs is required.

In photoacoustic imaging, the second near-infrared (NIR-II) window is where tissue generates the least background signal. However, the large size of the few available contrast agents in this spectral range impedes their pharmacokinetics and decreases their thermal stability, leading to unreliable photoacoustic imaging. Here, we report the synthesis of miniaturized gold nanorods absorbing in the NIR-II that are 5–11 times smaller than regular-sized gold nanorods with a similar aspect ratio. Under nanosecond pulsed laser illumination, small nanorods are about 3 times more thermally stable and generate 3.5 times stronger photoacoustic signal than their absorption-matched larger counterparts. These unexpected findings are confirmed using theoretical and numerical analysis, showing that photoacoustic signal is not only proportional to the optical absorption of the nanoparticle solution but also to the surface-to-volume ratio of the nanoparticles. In living tumour-bearing mice, these small targeted nanorods display a 30% improvement in efficiency of agent delivery to tumours and generate 4.5 times greater photoacoustic contrast. Miniaturized gold nanorods are reliable NIR-II photoacoustic agents, showing higher photothermal stability, enhanced photoacoustic signal and more efficient agent-to-tumour delivery compared with their larger counterparts.

Metastases, rather than primary tumors, determine mortality in the majority of cancer patients. A non-invasive immunofunctional imaging method was developed to detect sentinel lymph node (SLN) metastases using ultrasound-guided photoacoustic (USPA) imaging combined with glycol-chitosan-coated gold nanoparticles (GC-AuNPs) as an imaging contrast agent. GC-AuNPs, injected peritumorally into breast tumor-bearing mice, were taken up by immune cells, and subsequently transported to the SLN. Two-dimensional and three-dimensional USPA imaging was used to isolate the signal from GC-AuNP-tagged cells. Volumetric analysis was used to quantify GC-AuNP accumulation in the SLN after cellular uptake and transport by immune cells. The results show that the spatio-temporal distribution of GC-AuNPs in the SLN was affected by the presence of metastases. The parameter describing the spatial distribution of GC-AuNP-tagged cells within the SLN was more than 2-fold lower in metastatic lymph nodes compared with non-metastatic controls. Histological analysis confirmed that the distribution of GC-AuNP-tagged immune cells is changed by the presence of metastatic cells. The USPA immunofunctional imaging successfully distinguished metastatic from non-metastatic lymph nodes using biocompatible nanoparticles. This method could aid physicians in the detection of micrometastases, thus guiding SLN biopsy and avoiding unnecessary biopsy procedures.

Lymph node mapping is important in cancer immunotherapy because the morphology of lymph nodes is one of the crucial evaluation criteria of immune responses. We developed new theragnostic glycol-chitosan-coated gold nanoparticles (GC-AuNPs), which highlighted lymph nodes in ultrasound-guided photoacoustic (US/PA) imaging. Moreover, the ovalbumin epitope was conjugated GC-AuNPs (OVA-GC-AuNPs) for delivering tumor antigen to lymph node resident macrophage. In vitro studies proved the vigorous endocytosis activity of J774A.1 macrophage and consequent strong photoacoustic signals from them. The macrophages also presented a tumor antigen when OVA-GC-AuNPs were used for cellular uptake. After the lingual injection of GC-AuNPs into healthy mice, cervical lymph nodes were visible in a US/PA imaging system with high contrast. Three-dimensional analysis of lymph nodes revealed that the accumulation of GC-AuNPs in the lymph node increased as the post-injection time passed. Histological analysis showed GC-AuNPs or OVA-GC-AuNPs located in subcapsular and medullar sinuses where macrophages are abundant. Our new theragnostic GC-AuNPs present a superior performance in US/PA imaging of lymph nodes without targeting moieties or complex surface modification. Simultaneously, GC-AuNPs were able to deliver tumor antigens to cause macrophages to present the OVA epitope at targeted lymph nodes, which would be valuable for cancer immunotherapy.

Monoclonal antibodies (mAb) have had a transformative impact on treating cancers and immune-disorders. However, their use is limited by high development time and monetary cost, manufacturing complexities, suboptimal pharmacokinetics, and availability of disease-specific targets. To address some of these challenges, we developed an entirely synthetic, multivalent, Janus nanotherapeutic platform, called Synthetic Nanoparticle-Antibodies (SNAbs). SNAbs, with phage-display identified cell-targeting ligands on one “face”, and Fc-mimicking ligands on the opposite “face”, were synthesized using a custom, multi-step, solid-phase chemistry method. SNAbs efficiently targeted and depleted myeloid-derived immune-suppressor cells (MDSCs) from mouse-tumor and rat-trauma models, ex-vivo. Systemic injection of MDSC-targeting SNAbs efficiently depleted circulating MDSCs in a mouse triple-negative breast cancer model, enabling enhanced T cell and Natural Killer cell infiltration into tumors. Our results demonstrate that SNAbs are a versatile and effective functional alternative to mAbs, with advantages of a plug-and-play, cell-free manufacturing process, and high-throughput screening (HTS)-enabled library of potential targeting ligands.

Graphical abstract, In molecular and cellular photoacoustic imaging with exogenous contrast agents, image contrast is plagued by background resulting from endogenous absorbers in tissue. By using optically modulatable nanoparticles, we develop ultra-sensitive photoacoustic imaging by rejecting endogenous background signals and drastically improving signal contrast through time-delayed pump-probe pulsed laser illumination. Gated by prior pump excitation, modulatable photoacoustic (mPA) signals are recovered from unmodulatable background through simple, real-time image processing to yield background-free photoacoustic signal recovery within tissue mimicking phantoms and from ex-vivo tissues. Inherently multimodal, the fluorescence and mPA sensitivity improvements demonstrate the promise of Synchronously Amplified Photoacoustic Image Recovery (SAPhIRe) for PA imaging in diagnosis and therapy.

Early stage cancer detection technologies can provide functional information and potentially decrease the mortality rate caused by cervical cancer. In our previous work, a miniaturized ultrasound and photoacoustic endoscopic system has been developed to image the cervical tissue through the cervical canal to fulfills the need for a safe, low-cost, and high-resolution functional diagnostic system. However, the miniaturized size of endoscope and American National Standards Institute safety limits cause constraints of using high-intensity illumination during imaging. In addition, the strong light scattering of tissues limits the light penetration depth. Fortunately, the cervix anatomy allows for the delivery of additional light from the ectocervix by using an external illumination system. Here we propose a dual, co-planar illumination system, which can provide adequate illumination to the cervical tissue via combined internal and external light delivery strategies. Therefore, an increase in the area of light-tissue interaction allows us to raise the laser light energy while keeping fluence under safety limits. Thus, a reliable PA imaging can be obtained for the whole cervical tissue thickness. The system performance was tested using a Monte Carlo simulation, and laser-light fluence was calculated and compared at different depths within a simulated cervical-tissue model. The results indicated a higher and more uniform fluence in the Monte Carlo simulations. In addition, the photoacoustic imaging of the proposed system was evaluated by two cervical tissue-mimicking phantoms with human blood and graphite rods as inclusions inside it. In accordance with the simulations, the phantom study revealed a more reliable photoacoustic signal for the entire depth of the phantoms with an improved contrast to noise ratio and signal to noise ratio, and a higher coverage ratio of the imaging field of view. In summary, the dual-mode illumination system can provide more realistic information of inclusions within the tissue while considering safety limits, which can lead to more accuracy in biomarker detection for cervical cancer diagnostics.

Longitudinal monitoring of stem cells in the spinal cord could unveil critical information needed to understand regenerative processes, thereby expediting therapy development and translation. We introduce a post-operative trimodal imaging approach to monitor stem cells in the spinal cord over time. A key aspect of the approach is to label the stem cells with Prussian blue nanocubes (PBNCs), which simultaneously possess optical and magnetic properties for ultrasound-guided photoacoustic (US/PA) and magnetic resonance imaging (MRI) contrast. PBNC-Labeled stem cells were injected into the spinal cord of immunodeficient rats and tracked with US/PA imaging and MRI up to 14 d post-injection. Good agreement was observed between imaging modalities in vivo. Our results suggest that further development of the US/PA/MR imaging approach may create a powerful tool to aid development of regenerative therapies of the spinal cord, and the non-invasive imaging approach can ultimately be deployed in intra- and post-operative environments.

Two generation mechanisms—optical perturbation and acoustic radiation force (ARF)—were investigated where high frame rate ultrasound imaging was used to track the propagation of induced SAWs. We compared ARF-induced SAWs with laser-induced SAWs generated by laser beam irradiation of the uniformly absorbing tissue-like viscoelastic phantom, where light was preferentially absorbed at the surface. We also compared the frequency content of SAWs generated by ARF versus pulsed laser light, using the same duration of excitation. Differences in the SAW bandwidth were expected because, in general, laser light can be focused into a smaller area. Finally, we compared wave generation and propagation when the wave’s origin was below the surface. We also investigated the relationship between shear wave amplitude and optical fluence. The investigation reported here can potentially extend the applications of laser-induced SAW generation and imaging in life sciences and other applications.

An optical wavelength selection method based on the stability of the absorption cross-section matrix to improve spectroscopic photoacoustic (sPA) imaging was recently introduced. However, spatially-varying chromophore concentrations cause the wavelength- and depth-dependent variations of the optical fluence, which degrades the accuracy of quantitative sPA imaging. This study introduces a depth-optimized method that determines an optimal wavelength set minimizing an inverse of the multiplication of absorption cross-section matrix and fluence matrix to minimize the errors in concentration estimation. This method assumes that the optical fluence distribution is known or can be attained otherwise. We used a Monte Carlo simulation of light propagation in tissue with various depths and concentrations of deoxy-/oxy-hemoglobin. We quantitatively compared the developed and current approaches, indicating that the choice of wavelength is critical and our approach is effective especially when quantifying deeper imaging targets. Keywords: Spectroscopic photoacoustic imaging, Oxygen saturation estimation, Optimal wavelength selection, Spectral unmixing

A coherency of photoacoustic and ultrasound waves is dependent on the accuracy of the speedof- sound. This study evaluates the feasibility of a speed-of-sound estimation method for dual photoacoustic and ultrasound waves using laser-activated phase-change perfluorohexane nanodroplets (PFHnDs). The proposed method demonstrates that the estimated speed-of-sound (ESS) is close to the expected speed-of-sound for the medium (deviation = 0.3%), so it is useful for phase aberration correction in dual ultrasound and photoacoustic imaging.

Glaucoma is a major cause of blindness and is frequently associated with elevated intraocular pressure. The trabecular meshwork (TM), the tissue that primarily regulates intraocular pressure, is known to have reduced cellularity in glaucoma. Thus, stem cells, if properly delivered to the TM, may offer a novel therapeutic option for intraocular pressure control in glaucoma patients. For this purpose, targeted delivery of stem cells to the TM is desired. Here, we used magnetic nanoparticles (Prussian blue nanocubes [PBNCs]) to label mesenchymal stem cells and to magnetically steer them to the TM following injection into the eye’s anterior chamber. PBNC-labeled stem cells showed increased delivery to the TM vs. unlabeled cells after only 15-minute exposure to a magnetic field. Further, PBNC-labeled mesenchymal stem cells could be delivered to the entire circumference of the TM, which was not possible without magnetic steering. PBNCs did not affect mesenchymal stem cell viability or multipotency. We conclude that this labeling approach allows for targeted, relatively high-efficiency delivery of stem cells to the TM in clinically translatable time-scales, which are necessary steps towards regenerative medicine therapies for control of ocular hypertension in glaucoma patients.

Purpose This study introduces a real-time contrast-enhanced ultrasound imaging method with recently developed laser-activated nanodroplets (LANDs), a new class of phase-change nanometer-scale contrast agents that provides perceptible, sustained high-contrast with ultrasound. Methods In response to pulsed laser irradiation, the LANDs-, which contain liquid perfluorohexane and optical fuses-blink (vaporize and recondense). That is, they change their state from liquid nanodroplets to gas microbubbles, and then back to liquid nanodroplets. In their gaseous microbubble state, the LANDs provide high-contrast ultrasound, but the microbubbles formed in situ typically recondense in tens of milliseconds. As a result, LAND visualization by standard, real-time ultrasound is limited. However, the periodic optical triggering of LANDs allows us to observe corresponding transient, periodic changes in ultrasound contrast. This study formulates a probability function that measures how ultrasound temporal signals vary in periodicity. Then, the estimated probability is mapped onto a B-scan image to construct a LAND-localized, contrast-enhanced image. We verified our method through phantom and in vivo experiments using an ultrasound system (Vevo 2100, FUJIFILM VisualSonics, Inc., Toronto, ON, Canada) operating with a 40-MHz linear array and interfaced with a 10 Hz Nd:YAG laser (Phocus, Opotek Inc., Carlsbad, CA, USA) operating at the fundamental 1064 nm wavelength. Results From the phantom study, the results showed improvements in the contrast-to-noise ratio of our approach over conventional ultrasound ranging from 129% to 267%, with corresponding execution times of 0.10 to 0.29 s, meaning that the developed method is computationally efficient while yielding high-contrast ultrasound. Furthermore, in vivo sentinel lymph node (SLN) imaging results demonstrated that our technique could accurately identify the SLN. Conclusions The results indicate that our approach enables efficient and robust LAND localization in real time with substantially improved contrast, which is essential for the successful translation of this contrast agent platform to clinical settings.

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.

We present a gas-generating solid nanoparticle as a new concept of the ultrasound contrast agent. The developed nanoparticles are sufficiently small (less than 100 nm in diameter) to escape vasculature and yet, upon external pulsed laser light activation, release nitrogen gas for the enhanced contrast in ultrasound imaging. The gas-generating nanoconstructs combine the photocatalytic function of gold nanoparticles and photolysis of azide compounds. Using ultrasound imaging, we demonstrate the controlled, on-demand generation of nitrogen gas from nanoparticles due to the decomposition of azide groups trigggered by pulsed laser irradiation. The resulting gas forms bubbles that cause backscattered ultrasound signals and, therefore, modulate the contrast in ultrasound imaging.

Graphical abstract, Translation of stem cell therapies to treat injuries and diseases of the spinal cord is hindered by lack of real-time monitoring techniques to guide regenerative therapies intra- and postoperatively. Thus, we developed an ultrasound (US), photoacoustic (PA), and magnetic resonance (MR) imaging approach augmented with Prussian blue nanocubes (PBNCs) to guide stem cell injections intraoperatively and monitor stem cell therapies in the spinal cord postoperatively. Per the clinical procedure, a multi-level laminectomy was performed in rats ex vivo, and PBNC-labeled stem cells were injected directly into the spinal cord while US/PA images were acquired. US/PA/MR images were also acquired post-surgery. Several features of the imaging approach were demonstrated including detection of low stem cell concentrations, real-time needle guidance and feedback on stem cell delivery, and good agreement between US/PA/MR images. These benefits span intra- and postoperative environments to support future development of this imaging tool.

Laser-activated perfluorocarbon nanodroplets are an emerging class of phase change, dual-contrast agents that can be utilized in ultrasound and photoacoustic imaging. Through the ability to differentiate subpopulations of nanodroplets via laser activation at different wavelengths of near-infrared light, optically-triggered color-coded perfluorocarbon nanodroplets present themselves as an attractive tool for multiplexed ultrasound and photoacoustic imaging. In particular, laser-activated droplets can be used to provide quantitative spatiotemporal information regarding distinct biological targets, allowing for their potential use in a wide range of diagnos tic and therapeutic applications. In the work presented, laser-activated color-coded perfluorocarbon nanodroplets are synthesized to selectively respond to laser irradiation at corresponding wavelengths. The dynamic ultrasound and photoacoustic signals produced by laser-activated perfluorocarbon nanodroplets are evaluated in situ prior to implementation in a murine model. In vivo, these particles are used to distinguish unique particle trafficking mechanisms and are shown to provide ultrasound and photoacoustic contrast for up to 72 hours within lymphatics. Overall, the conducted studies show that laser-activated color-coded perfluorocarbon nanodroplets are a promising agent for multiplexed ultrasound and photoacoustic imaging.

The protective barriers of the CNS present challenges during the treatment and monitoring of diseases. In particular, the blood brain barrier is a major hindrance to the delivery of imaging contrast agents and therapeutics to the brain. In this work, we use gas microbubble-assisted focused ultrasound to transiently open the blood brain barrier and locally deliver silica coated gold nanorods across the barrier. This particular nanoagent possesses a strong optical absorption which enables in vivo and ex vivo visualization of the delivered particles using ultrasound-guided photoacoustic imaging. The results of these studies demonstrate the potential of ultrasound-guided photoacoustics to image contrast agents delivered via microbubble-assisted focused ultrasound for longitudinal diagnostic imaging and for therapeutic monitoring of neurological diseases.

Significance: Stem cell therapies are of interest for treating a variety of neurodegenerative diseases and injuries of the spinal cord. However, the lack of techniques for longitudinal monitoring of stem cell therapy progression is inhibiting clinical translation. Aim: The goal of this study is to demonstrate an intraoperative imaging approach to guide stem cell injection to the spinal cord in vivo. Results may ultimately support the development of an imaging tool that spans intra- or postoperative environments to guide therapy throughout treatment. Approach: Stem cells were labeled with Prussian blue nanocubes (PBNCs) to facilitate combined ultrasound and photoacoustic (US/PA) imaging to visualize stem cell injection and delivery to the spinal cord in vivo. US/PA results were confirmed by magnetic resonance imaging (MRI) and histology. Results: Real-time intraoperative US/PA image-guided injection of PBNC-labeled stem cells and three-dimensional volumetric images of injection provided feedback necessary for successful delivery of therapeutics into the spinal cord. Postoperative MRI confirmed delivery of PBNC-labeled stem cells. Conclusions: The nanoparticle-augmented US/PA approach successfully detected injection and delivery of stem cells into the spinal cord, confirmed by MRI. Our work demonstrated in vivo feasibility, which is a critical step toward the development of a US/PA/MRI platform to monitor regenerative spinal cord therapies.

Nanoparticles play an important role in biomedicine. We have developed a method for size-controlled synthesis of photomagnetic Prussian blue nanocubes (PBNCs) using superparamagnetic iron oxide nanoparticles (SPIONs) as precursors. The developed PBNCs have magnetic and optical properties desired in many biomedical diagnostic and therapeutic applications. Specifically, the size-tunable photomagnetic PBNCs exhibit high magnetic saturation, strong optical absorption with a peak at approximately 700 nm, and superior photostability. Our studies demonstrate that PBNCs can be used as MRI and photoacoustic imaging contrast agents in vivo. We also showed the utility of PBNCs for labeling and magnetic manipulation of cells. Dual magnetic and optical properties, together with excellent biocompatibility, render PBNCs an attractive contrast agent for both diagnostic and therapeutic applications. The use SPIONs as precursors for PBNCs provides flexibility and allows researchers to design theranostic agents according to required particle size, optical, and magnetic properties.

Combined ultrasound and photoacoustic imaging systems are being developed for biomedical and clinical applications. One common probe configuration is to use a linear transducer array with external light delivery to produce coregistered ultrasound and photoacoustic images. The diagnostic capability of these systems is dependent on the effectiveness of light delivery to the imaging target. We use Monte Carlo modeling to investigate the optimal design geometry of an integrated probe. Simulations are conducted with multiple tissue compositions and wavelengths. The effect of a skin layer with the thickness of a mouse or a human is also considered. The model was validated using a tissue-mimicking gelatin phantom and corresponding Monte Carlo simulations. The optimal illumination angle is shallower with human skin thickness, whereas intermediate angles are ideal with mouse skin thickness. The effect of skin thickness explains differences in the results of prior work. The simulations also indicate that even with identical hardware and imaging parameters, light delivery will be up to 3× smaller in humans than in mice, due to the increased scattering from thicker skin. Our findings have clear implications for the many researchers using mice to test and develop imaging methods for clinical translation.

This study explores photoacoustic (PA) speckle tracking to characterize flow as an alternative to ultrasound (US) speckle tracking or current PA flow imaging methods. In cases where tracking of submicrometer particles is required, the US signal-to-noise ratio and contrast might be low due to limited reflectivity of subwavelength size targets at low concentrations. However, it may be possible to perform more accurate velocimetry using PAs due to different contrast mechanisms utilized in PA imaging. Here, we introduce a PA-based speckle tracking method that overcomes the directional dependence of Doppler imaging and the limited field of view of current correlation-based methods used in PA flow imaging. The feasibility of this method is demonstrated in a potential application-minimally invasive diagnosis of ventricular shunt malfunction, where the velocity of optically absorbing particles was estimated in a shunt catheter using block matching of PA and US signals. Overall, our study demonstrates the potential of the PA-based motion tracking method under various flow rates where US imaging cannot be effectively used for specking tracking because of its low contrast and low signal-to-noise ratio.

Clinical imaging techniques for the anterior segment of the eye provide excellent anatomical information, but molecular imaging techniques are lacking. Molecular photoacoustic imaging is one option to address this need, but implementation requires use of contrast agents to distinguish molecular targets from background photoacoustic signals. Contrast agents are typically selected based on a priori knowledge of photoacoustic properties of tissues. However, photoacoustic properties of anterior ocular tissues have not been studied yet. Herein, anterior segment anatomy and corresponding photoacoustic signals were analyzed in brown and blue porcine eyes ex vivo. Measured photoacoustic spectra were compared to known optical absorption spectra of endogenous chromophores. In general, experimentally measured photoacoustic spectra matched expectations based on absorption spectra of endogenous chromophores reported in the literature, and similar photoacoustic spectra were observed in blue and brown porcine eyes. However, unique light–tissue interactions at the iris modified photoacoustic signals from melanin. Finally, we demonstrated how the measured PA spectra established herein can be used for one application of molecular PA imaging, detecting photoacoustically labeled stem cells in the anterior segment for glaucoma treatment.

Super-resolution imaging with moving microbubbles has shown potential in identifying fine details of deeplying vascular compartments. To image the extravascular targets, the current study has employed nanometer-sized, optically triggered perfluorohexane nanodroplets (PFHnDs). In response to pulsed laser irradiation, the PFHnDs repeatedly vaporize and stochastically recondense, resulting in random changes of ultrasound signals. Our previous study has shown that the stochastic recondensation of the PFHnDs can be used to isolate individual PFHnDs for super-resolution imaging. This study introduces an improved method for super-resolution imaging with ultrafast ultrasound imaging of PFHnDs. The previous method was based on subtraction of two consecutive ultrasound images to detect signals from recondensed, isolated droplets whereas our current method compounds respective multiple pre- and post-recondensation ultrafast ultrasound images prior to subtraction to improve the spatial resolution further. To evaluate the axial and lateral resolutions of our method, we repeatedly imaged a phantom containing PFHnDs using a programmable ultrasound system synchronized with a pulsed laser system. As a result, our method improved the lateral and axial resolutions by 54% and 68%, respectively, over the previous super-resolution imaging approach, indicating that it can be used for localizing extravascular molecular targets with superior accuracy.

Objective: The purpose of this study was to introduce combined multiwavelength photoacoustic (PA) and plane-wave ultrasound (US) imaging referred to as mwPA/pwUS imaging capable of probing the rapid dynamic behavior of optically activated phase-change contrast agents. Methods: A dedicated mwPA/pwUS imaging sequence was developed based on a programmable US system synchronized with a tunable laser to irradiate tissue with laser pulses at desired optical wavelengths and to acquire post laser pulse PA images followed by ultrafast plane-wave US images. To evaluate the mwPA/pwUS imaging, a capillary filled with optically responsive perfluorohexane nanodroplets (PFHnDs) containing a dye with the peak absorption at 760 nm was imaged with optical wavelengths ranging from 700 to 940 nm. The differences between post-laser ultrafast US images [i.e., differential US (ΔUS)] were taken to visualize the recondensation dynamics of PFHnDs at each wavelength. Results: The PA images of PFHnDs showed higher contrast near 760 nm wavelength, corresponding to the peak absorption of the dye encapsulated in the PFHnDs. Moreover, the ΔUS signals immediately after 760-nm pulsed laser irradiation were also high due to the increased US contrast associated with vaporized PFHnDs. Conclusion: The mwPA/pwUS imaging allowed for the US-based optical spectroscopic characterization of PFHnDs and their dynamics. Significance: The introduced mwPA/pwUS imaging sequence can be used in various clinical applications where both spectroscopic PA imaging of endogenous and/or exogenous chromophores and ultrafast US imaging of phase-change nanodroplets are desired.

Accurate non-invasive assessment of tissue elasticity in vivo is required for early diagnostics of many tissue abnormalities. We have developed a focused air-pulse system that produces a low-pressure and short-duration air stream, which can be used to excite transient surface waves (SWs) in soft tissues. System characteristics were studied using a high-resolution analog pressure transducer to describe the excitation pressure. Results indicate that the excitation pressure provided by the air-pulse system can be easily controlled by the air source pressure, the angle of delivery, and the distance between the tissue surface and the port of the air-pulse system. Furthermore, we integrated this focused air-pulse system with phase-sensitive optical coherence tomography (PhS-OCT) to make non-contact measurements of tissue elasticity. The PhS-OCT system is used to assess the group velocity of SW propagation, which can be used to determine Young's modulus. Pilot experiments were performed on gelatin phantoms with different concentrations (10%, 12% and 14% w/w). The results demonstrate the feasibility of using this focused air-pulse system combined with PhS-OCT to estimate tissue elasticity. This easily controlled non-contact technique is potentially useful to study the biomechanical properties of ocular and other tissues in vivo.

OBJECT Cerebral shunt malfunction is common but often difficult to effectively diagnose. Current methods are invasive, involve ionizing radiation, and can be costly. The authors of this study investigated the feasibility of quantitatively measuring CSF flow in a shunt catheter using contrast-enhanced ultrasound. METHODS A syringe pump was used to push a solution of gas-filled microbubbles at specific flow rates through a shunt catheter while a high-frequency ultrasound imaging system was used to collect ultrasound images for offline processing. Displacement maps and velocity profiles were generated using a speckle-tracking method based on a cross-correlation algorithm. An additional correction factor, to account for a predictable underestimation and to adjust the measured flow rates, was calculated based on the geometry of the ultrasound imaging plane and assuming a simple model of laminar flow. RESULTS The developed method was able to differentiate between physiologically relevant flow rates, including no flow and 0.006 to 0.09 ml/min, with reasonable certainty. The quantitative measurement of flow rates through the catheter using this method was determined to be in good agreement with the expected flow rate. CONCLUSIONS This study demonstrated that contrast-enhanced ultrasound has the potential to be used as a minimally invasive and cost-effective alternative method for outpatient shunt malfunction diagnosis.

To determine the ability of ultrasonography (US)-guided spectroscopic photoacoustic (sPA) imaging to depict changes in blood oxygen saturation (SO2) in metastatic lymph nodes of a mouse model of oral cancer.All studies were performed by following protocols approved by the institutional animal care and use committee at the University of Texas at Austin. Coregistered US and photoacoustic images were acquired spanning volumes containing a total of 31 lymph nodes in 17 female nu/nu mice. The mice were either healthy (three mice, five nodes) or bearing a primary tumor consisting of luciferase-labeled FaDu cells (14 mice, 26 nodes). Ten photoacoustic images acquired with optical wavelengths spanning from 680 to 860 nm were spectrally unmixed by using a linear least-squares method to obtain sPA images. After imaging, histologic analysis enabled confirmation of the presence of micrometastases. Generalized estimating equations were used to compare metastatic and normal lymph nodes, with a P value of .05 taken to indicate a significant difference. Sensitivity and specificity were determined with a receiver operator characteristic curve constructed from the background-subtracted SO2 values.Metastatic lymph nodes (n = 7) exhibited a significantly (P = .018) lower spatially averaged background-subtracted SO2 (mean, 5.4% ± 3.5 [standard error]) when compared with lymph nodes without metastases (mean, 13.7% ± 1.3; n = 24). This effect was observed throughout the entire volume of the nodes rather than being limited to the metastatic foci. The change in SO2, which was inversely related to the size of the metastasis, was detectable in metastases as small as 2.6 × 10(-3) mm(3).The results show that US-guided sPA imaging is capable of depicting changes in SO2 in lymph nodes that were correlated with metastatic invasion.

Many biomedical applications necessitate a targeted intracellular delivery of the nanomaterial to specific cells. Therefore, a non-invasive and reliable imaging tool is required to detect both the delivery and cellular endocytosis of the nanoparticles. Herein, we demonstrate that magneto-photo-acoustic (MPA) imaging can be used to monitor the delivery and to identify endocytosis of magnetic and optically absorbing nanoparticles. The relationship between photoacoustic (PA) and magneto-motive ultrasound (MMUS) signals from the in vitro samples were analyzed to identify the delivery and endocytosis of nanoparticles. The results indicated that during the delivery of nanoparticles to the vicinity of the cells, both PA and MMUS signals are almost linearly proportional. However, accumulation of nanoparticles within the cells leads to nonlinear MMUS-PA relationship, due to non-linear MMUS signal amplification. Therefore, through longitudinal MPA imaging, it is possible to monitor the delivery of nanoparticles and identify the endocytosis of the nanoparticles by living cells.

In this study the dynamic behavior of a layered viscoelastic medium in response to the harmonic and impulsive acoustic radiation force applied to its surface was investigated both theoretically and experimentally. An analytical solution for a layered viscoelastic compressible medium in frequency and time domains was obtained using the Hankel transform. A special incompressible case was considered to model soft biological tissues. To verify our theoretical model, experiments were performed using tissue-like gel-based phantoms with varying mechanical properties. A 3.5 MHz single-element focused ultrasound transducer was used to apply the radiation force at the surface of the phantoms. A phase-sensitive optical coherence tomography (OCT) system was used to track the displacements of the phantom surface. Theoretically predicted displacements were compared with experimental measurements. The role of the depth dependence of the elastic properties of a medium in its response to an acoustic pulse at the surface was studied. It was shown that the low-frequency vibrations at the surface are more sensitive to the deep layers than high-frequency ones. Therefore, the proposed model in combination with spectral analysis can be used to evaluate depth-dependent distribution of the mechanical properties based on the measurements of the surface deformation.

PURPOSE To evaluate the capability of a novel, coaligned focused ultrasound and phase-sensitive optical coherence elastography (US-OCE) system to assess age-related changes in biomechanical properties of the crystalline lens in situ. METHODS Low-amplitude elastic deformations in young and mature rabbit lenses were measured by an US-OCE system consisting of a spectral-domain optical coherence tomography (OCT) system coaligned with a focused ultrasound system used to produce a transient force on the lens surface. Uniaxial compressional tests were used to validate the OCE data. RESULTS The OCE measurements showed that the maximum displacements of the young rabbit lenses were significantly larger than those of the mature lenses, indicating a gradual increase of the lens stiffness with age. Temporal analyses of the displacements also demonstrate a similar trend of elastic properties in these lenses. The stress-strain measurements using uniaxial mechanical tests confirmed the results obtained by the US-OCE system. CONCLUSIONS The results demonstrate that the US-OCE system can be used for noninvasive analysis and quantification of lens biomechanical properties in situ and possibly in vivo.

Shear wave elasticity imaging (SWEI) enables the quantitative assessment of the mechanical properties of tissue. In SWEI, the effective generation of acoustic radiation force is of paramount importance. Consequently, several research groups have investigated various transmit beamforming and pulse-sequencing methods. To further improve the efficiency of shear wave generation, and therefore, to increase the quality of SWEI, we introduce a technique referred to as “multi-angle compound SWEI” (MAC-SWEI), which uses simultaneous multi-angular push beams created by dual-phase transmit focusing. By applying a constant phase offset on every other element of an array transducer, dual-phase transmit focusing creates both main and grating lobes (i.e., multi-angular push beams for pushing) to simultaneously generate shear waves with several wave-front angles. The shear waves propagating at different angles are separated by multi-directional filtering in the frequency domain, leading to the reconstruction of multiple spatially co-registered shear-wave velocity maps. To form a single elasticity image, these maps are combined, while regions associated with known artifacts created by the push beams are omitted. Overall, we developed and tested the MAC-SWEI method using Field II quantitative simulations and the experiments performed using a programmable ultrasound imaging system. Our results suggest that MAC-SWEI with dual-phase transmit focusing may improve the quality of elasticity maps.

Contrast-enhanced ultrasound imaging utilizing the nonlinear responses of microbubbles is proposed for identifying ventricular shunt malfunction. The developed method suppresses the signal from walls of a shunt catheter and background tissues, and allows accurate measurements of the cerebrospinal fluid flow within the shunt catheter using a relatively small concentration of microbubbles. The flow rates estimated in the linear mode were significantly underestimated (65% at 0.1 ml/min and 5.0% microbubble concentration) while estimates using the nonlinear mode were not. Overall, the nonlinear responses of microbubbles improve the estimation of flow rates in a shunt catheter at low concentrations of microbubbles.

We hypothesize that dissipation of mechanical energy of external impact to absorb mechanical shock is a fundamental function of skeletal muscle in addition to its primary function to convert chemical energy into mechanical energy. In physical systems, the common mechanism for absorbing mechanical shock is achieved with the use of both elastic and viscous elements and we hypothesize that the viscosity of the skeletal muscle is a variable parameter which can be voluntarily controlled by changing the tension of the contracting muscle. We further hypothesize that an ability of muscle to absorb shock has been an important factor in biological evolution, allowing the life to move from the ocean to land, from hydrodynamic to aerodynamic environment with dramatically different loading conditions for musculoskeletal system. The ability of muscle to redistribute the energy of mechanical shock in time and space and unload skeletal joints is of key importance in physical activities. We developed a mathematical model explaining the absorption of mechanical shock energy due to the increased viscosity of contracting skeletal muscles. The developed model, based on the classical theory of sliding filaments, demonstrates that the increased muscle viscosity is a result of the time delay (or phase shift) between the mechanical impact and the attachment/detachment of myosin heads to binding sites on the actin filaments. The increase in the contracted muscle’s viscosity is time dependent. Since the forward and backward rate constants for binding the myosin heads to the actin filaments are on the order of 100 s 1 , the viscosity of the contracted muscle starts to significantly increase with an impact time greater than 0.01 s. The impact time is one of the key parameters in generating destructive stress in the colliding objects. In order to successfully dampen a short high power impact, muscles must first slow it down to engage the molecular mechanism of muscle viscosity. Muscle carries out two functions, acting first as a nonlinear spring to slow down impact and second as a viscous damper to absorb the impact. Exploring the ability of muscle to absorb mechanical shock may shed light to many problems of medical biomechanics and sports medicine. Currently there are no clinical devices for real-time quantitative assessment of viscoelastic properties of contracting muscles in vivo. Such assessment may be important for diagnosis and monitoring of treatment of various muscle disorders such as muscle dystrophy, motor neuron diseases, inflammatory and metabolic myopathies and many more.

By mapping the distribution of targeted plasmonic nanoparticles (NPs), photoacoustic (PA) imaging offers the potential to detect the pathologies in the early stages. However, optical absorption of the endogenous chromophores in the background tissue significantly reduces the contrast resolution of photoacoustic imaging. Previously, we introduced MPA imaging – a synergistic combination of magneto-motive ultrasound (MMUS) and PA imaging, and demonstrated MPA contrast enhancement using cell culture studies. In the current study, contrast enhancement was investigated in vivo using the magneto-photo-acoustic (MPA) imaging augmented with dual-contrast nanoparticles. Liposomal nanoparticles (LNPs) possessing both optical absorption and magnetic properties were injected into a murine tumor model. First, photoacoustic signals were generated from both the endogenous absorbers in the tissue and the liposomal nanoparticles in the tumor. Then, given significant differences in magnetic properties of tissue and LNPs, the magnetic response of LNPs (i.e. MMUS signal) was utilized to suppress the unwanted PA signals from the background tissue thus improving the PA imaging contrast. In this study, we demonstrated the 3D MPA imaging of LNP-labeled xenografted tumor in a live animal. Compared to conventional PA imaging, the MPA imaging show significantly enhanced contrast between the nanoparticle-labeled tumor and the background tissue. Our results suggest the feasibility of MPA imaging for high contrast in vivo mapping of dual-contrast nanoparticles.

In nanoparticle-augmented photothermal therapy, evaluating the delivery and spatial distribution of nanoparticles, followed by remote temperature mapping and monitoring, is essential to ensure the optimal therapeutic outcome. The utility of ultrasound and photoacoustic imaging to assist photothermal therapy has been previously demonstrated. Here, using a mouse xenograft tumor model, it is demonstrated in vivo that ultrasound-guided photoacoustic imaging can be used to plan the treatment and to guide the therapy. To evaluate nanoparticle delivery and spatial distribution, three-dimensional ultrasound and spectroscopic photoacoustic imaging of a mouse with a tumor was performed before and after intravenous injection of silica-coated gold nanorods. After injection and sufficient circulation of nanoparticles, photothermal therapy was performed for 5 min using an 808-nm continuous-wave laser. During the photothermal therapy, photoacoustic images were acquired continuously and used to measure the temperature changes within tissue. A heterogeneous distribution of temperature, which was spatially correlated with the measured distribution of nanoparticles, indicated that peak temperatures of 53°C were achieved in the tumor. An Arrhenius thermal damage model determined that this thermal deposition would result in significant cell death. The results of this study suggest that ultrasound and photoacoustic imaging can effectively guide photothermal therapy to achieve the desired thermal treatment.

Although various ophthalmic imaging methods, including fundus photography and optical coherence tomography, have been applied for effective diagnosis of ocular diseases with high spatial resolution, most of them are limited by shallow imaging penetration depth and a narrow field of view. Also, many of those imaging modalities are optimized to provide microscopic anatomical information, while functional or cellular information is lacking. Compared to other ocular imaging modalities, photoacoustic imaging can achieve relatively deep penetration depth and provide more detailed functional and cellular data based on photoacoustic signal generation from endogenous contrast agents such as hemoglobin and melanin. In this paper, array-based ultrasound and photoacoustic imaging was demonstrated to visualize pigmentation in the eye as well as overall ocular structure. Fresh porcine eyes were visualized using a real-time ultrasound micro-imaging system and an imaging probe supporting laser pulse delivery. In addition, limited photoacoustic imaging field of view was improved by an imaging probe tilting method, enabling visualization of most regions of the retina covered in the ultrasound imaging.