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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.

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

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.

The high mortality rate of cervical cancer can be minimized by early-stage cancer detection or providing functional information about the tissue to provide an effective treatment. Such clinical procedures require the need for a safe, low-cost, and high-resolution functional diagnostic system. Previously, we proposed a dual-modal ultrasound and photoacoustic imaging endoscope with a miniaturized footprint for imaging the cervical tissue through the cervical canal. Since the light delivery system in our endoscope can carry a limited amount of light fluence (restricted to ANSI safety limits), we proposed to develop a dual, co-planar illumination system to irradiate cervical tissue both from inside and outside. The proposed endoscopic system was developed by coupling the external illumination system to the developed US/PA imaging endoscope. The external illumination system can provide additional illumination to the cervical tissue through the vaginal fornix area. The total diameter of the external illumination system is 4.2 mm and comprises of seven optical fibers. The performance of the system was evaluated through a tissue-mimicking phantom. Results indicated that the combined dual-illumination system enabled PA imaging to provide a higher consistent signal amplitude. In addition, an enhanced signal to noise ratio and contrast to noise ratio was achieved throughout the whole mimicked tissue phantom thickness while complying with the safety limits for light fluence.

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.

We present the results on development of the 3D imaging platform combining photoacoustic tomography and fluorescence (PAFT) for preclinical and biological research. This combined multimodal imaging instrument addresses known deficiencies in sensitivity, spatial resolution, and anatomical registration of the individual imaging components. Multiangle photoacoustic projections, excited by an OPO operating in the near-infrared window, of a live anesthetized animal are used to reconstruct large volumes (30 cm3) that show deep anatomical vasculature and blood-rich tissues with resolutions exceeding 150 μm. A sCMOS camera is used for simultaneous co-registered multi-angle optical imaging. The images of a fluorescent dual-contrast agent are then reconstructed into a 3D volume using a tomographic algorithm. A separate 532-nm low-energy pulsed laser excitation is used for skin topography and imaging of superficial vasculature. All three imaging channels can be combined to produce spatially accurate in vivo volumes showing an animal’s skin, deep anatomical structures, and distribution of photosensitive molecular contrast agents. PAFT’s photoacoustic sensitivity was assessed using contrast agents in a phantom study. We demonstrate biomedical imaging application of PAFT’s combined imaging modalities by observing biodistribution of a dual-contrast agent injected intravenously to in vivo preclinical murine models.

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

OBJECTIVES Intravascular photoacoustic (IVPA) imaging is being developed to image atherosclerotic plaques, a leading cause of morbidity and mortality in the United States. However, the safety of this imaging modality, which requires repeated irradiation with short laser pulses, has not yet been investigated. This study has two objectives. First, determine in vitro the limit of cumulative fluence that can be applied to cells before death at IVPA relevant wavelengths. Second, evaluate if high single pulse fluences are a potential cause of cell death during IVPA imaging. MATERIALS AND METHODS Experiments were conducted using endothelial cells, macrophages, and smooth muscle cells. The cumulative fluence experiments were conducted at 1064 and 1197 nm, using a high pulse repetition frequency laser. Cells were irradiated with a wide range of cumulative fluences and evaluated for cell death. The thresholds for death were compared to the maximum expected clinical cumulative fluence. To evaluate the effect of single pulse fluences, cells were irradiated at 1064, 1210, and 1720 nm. Light was delivered at a range of pulse energies to emulate the fluences that cells would be exposed to during clinical IVPA imaging. RESULTS At 1064 nm, all three cell types remained viable at cumulative fluences above the maximum expected clinical cumulative fluence, which is calculated based on common IVPA imaging protocols. At 1197 nm, cells were viable near or just below the maximum expected clinical cumulative fluence, with some cell type to cell type variation. All three cell types remained viable after irradiation with high single pulse fluences at all three wavelengths. CONCLUSION The cumulative fluence experiments indicate that safety considerations are likely to put constraints on the amount of irradiation that can be used in IVPA imaging protocols. However, this study also indicates that it will be possible to use IVPA imaging safely, since cumulative fluences could be reduced by as much as two orders of magnitude below the maximum expected clinical cumulative fluence by varying the imaging protocol, albeit at the expense of image quality. The single pulse fluence experiments indicate that cell death from single pulse fluence is not likely during IVPA imaging. Thus, future studies should focus on heat accumulation as the likely mechanism of tissue damage. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

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.

Regenerative therapies using stem cells have great potential for treating neurodegenerative diseases and traumatic injuries in the spinal cord. In spite of significant research efforts, many therapies fail at the clinical phase. As stem cell technologies advance toward clinical use, there is a need for a minimally invasive, safe, affordable, and real-time imaging technique that allows for the accurate and safe monitoring of stem cell delivery in the operating room. In this work, we present a combined ultrasound and photoacoustic imaging tool to provide image-guided needle placement and monitoring of nanoparticle-labeled stem cell delivery into the spinal cord. We successfully tagged stem cells using gold nanospheres and provided image-guided delivery of stem cells into the spinal cord in real-time, detecting as few as 1000 cells. Ultrasound and photoacoustic imaging was used to guide needle placement for direct stem cell injection to minimize the risk of needle shear and accidental injury and to improve therapeutic outcomes with accurate, localized stem cell delivery. Following injections of various volumes of cells, three-dimensional ultrasound and photoacoustic images allowed the visualization of stem cell distribution along the spinal cord, showing the potential to monitor the migration of the cells in the future. The feasibility of quantitative imaging was also shown by correlating the total photoacoustic signal over the imaging volume to the volume of cells injected. Overall, the presented method may allow clinicians to utilize imaged-guided delivery for more accurate and safer stem cell delivery to the spinal cord.

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.

Stem cell therapies are a promising treatment for many patients suffering from diseases with poor prognosis. However, clinical translation is inhibited by a lack of in vivo monitoring techniques to...

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.

We report on the development of a preclinical 3D imaging platform integrating photoacoustic tomography and fluorescence (PAFT). The proposed multimodal imaging concept addresses known deficiencies in sensitivity, anatomical registration, and spatial resolution of the individual imaging modalities. Multi-view photoacoustic and optical projections of the studied animal are utilized to reconstruct large (27 cm3) volumes showing vascular network and blood-rich tissues, as well as regions with induced optical/fluorescence contrast with 3D resolution exceeding 150 μm. An additional 532-nm low-energy pulsed laser excitation is implemented as a separate imaging channel for registration over skin topography and superficial vasculature. PAFT technology enables functional and molecular volumetric imaging using wide range of fluorescent and luminescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures of the studied animal model. We demonstrated the PAFT performance using phantoms and by in vivo imaging of preclinical murine models.

Pulsed laser irradiation has emerged as an effective means to photothermally transform plasmonic nanostructures after their use in different biomedical applications. However, the ability to predict the products after photothermal transformation requires extensive ex situ studies. Here, we report a systematic study of the photothermal transformation of Au-Ag nanocages with a localized surface plasmon resonance at ca. 750 nm under pulsed laser irradiation at different fluences and a pulse duration of 5 ns. At biologically relevant laser energies, the pulsed laser transforms Au-Ag nanocages into pseudo-spherical, solid nanoparticles. The solid nanoparticles contained similar numbers of Au and Ag atoms to the parent Au-Ag nanocages. At increased laser fluences (>16 mJ cm-2) and number of pulses (>150), the average diameter of the resulting pseudo-spherical particles increased due to the involvement of Ostwald ripening and/or attachment-based growth. The changes in optical properties as a result of the transformation were validated using simulations based on the discrete dipole approximation method, where the spectral profiles and peak positions of the initial and final states matched well with the experimentally derived data. The results may have implications for the future use of Au-Ag nanocages in biomedicine, catalysis, and sensing.

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.

Photoacoustics has emerged as a high-contrast imaging modality that provides optical absorption maps inside of tissues, therefore complementing morphological information of conventional ultrasound. The laser-generated photoacoustic waves are usually envelope-detected, thus disregarding the specific waveforms generated by each photoabsorber. Here we propose a sparsity-promoting image reconstruction method that allows the estimation of each photoabsorber's source-time function. Preliminary studies showed the ability to reconstruct the optical absorption map of an in silico vessel phantom. By using a sparsity-promoting imaging method, absorption maps and source-time functions can still be recovered even in situations where the number of transducers is decreased by a factor of six. Moreover, the recovery is able to attain higher resolution than conventional beamforming methods. Because our method recovers the source-time function of the absorbers, it could potentially also be used to distinguish different types of photoabsorbers, or the degree of aggregation of exogenous agents, under the assumption that these would generate different source-time functions at the moment of laser irradiation.

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.

Photoacoustic imaging using exogenous contrast agents has emerged as a hybrid technique that enables the deep imaging of optical properties of tissues with high spatial resolution. The power of this imaging technique can be greatly enhanced by the use of contrast agents that absorb at near-infrared wavelengths and whose optical properties can be modulated in response to the local environment. We have designed contrast agents consisting of gold nanoparticles coated with anisotropic silica nanoshells. The tunable aggregation of these janus particles in cell culture media resulted in a dramatic amplification of photoacoustic signals in the near-infrared region. We also demonstrated imaging using these contrast agents in mammalian cells, including macrophages and breast cancer cells as well as in vivo. The ability to modulate janus particle aggregation in response to a range of stimuli in combination with the high resolution and deep penetration of multiwavelength photoacoustic imaging are attractive for a broad range of applications in diagnostic imaging and theranostics.

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.

We have developed a preclinical 3D imaging instrument integrating photoacoustic tomography and fluorescence (PAFT) addressing known deficiencies in sensitivity and spatial resolution of the individual imaging components. PAFT is designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct registration of the two imaging modalities. Orthogonal photoacoustic projections are utilized to reconstruct large (21 cm3 ) volumes showing vascularized anatomical structures and regions of induced optical contrast with spatial resolution exceeding 100 µm. The major advantage of orthogonal fluorescence projections is significant reduction of background noise associated with transmitted or backscattered photons. The fluorescence imaging component of PAFT is used to boost detection sensitivity by providing low-resolution spatial constraint for the fluorescent biomarkers. PAFT performance characteristics were assessed by imaging optical and fluorescent contrast agents in tissue mimicking phantoms and in vivo. The proposed PAFT technology will enable functional and molecular volumetric imaging using fluorescent biomarkers, nanoparticles, and other photosensitive constructs mapped with high fidelity over robust anatomical structures, such as skin, central and peripheral vasculature, and internal organs.