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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.

To overcome the most significant deficiencies of conventional and contrast-enhanced ultrasound imaging—low contrast and large size of microbubbles, we introduced new class of contrast agents—nanometer scale particles that are capable of escaping vascular compartments, penetrating into tissue, and then, once they reach the target site, generating sufficient ultrasound and photoacoustic contrast upon user-controlled optical activation. These multimodal contrast agents—phase-change perfluorocarbon nanodroplets and plasmonic nanoparticles covered by azide compounds—are stable at physiological temperatures, biocompatible, and monodisperse in size. Given the unique properties of the particles, our approach to image these particles is drastically different and is based on ultrasound read-out of the optically induced temporal changes. Specifically, time-varying ultrasound signals exhibited by the nanoparticles versus the static background are used to reconstruct a high contrast, background-free image of the contrast agent. Furthermore, these particles allow for multiplexed molecular imaging by permitting user-controlled triggering of distinct color-coded populations of contrast agents via tuning of the incident laser irradiation to match peak optical absorption of the particles. Finally, nanoparticles may also contain therapeutic cargo and thus can be used for controlled drug delivery and release. This presentation, via examples, will discuss diagnostic imaging and image-guided therapy using the gas-generating nanoparticles.

An approach to assess the mechanical properties of a viscoelastic medium using laser-induced microbubbles is presented. To measure mechanical properties of the medium, dynamics of a laser-induced cavitation microbubble in viscoelastic medium under acoustic radiation force was investigated. An objective lens with a 1.13 numerical aperture and an 8.0 mm working distance was designed to focus a 532 nm wavelength nanosecond pulsed laser beam and to create a microbubble at the desired location. A 3.5 MHz ultrasound transducer was used to generate acoustic radiation force to excite a laser-induced microbubble. Motion of the microbubble was tracked using a 25 MHz imaging transducer. Agreement between a theoretical model of bubble motion in a viscoelastic medium and experimental measurements was demonstrated. Young’s modulii reconstructed using the laser-induced microbubble approach were compared with those measured using a direct uniaxial method over the range from 0.8 to 13 kPa. The results indicate good agreement between methods. Thus, the proposed approach can be used to assess the mechanical properties of a viscoelastic medium.

Diagnostic ultrasound imaging often relies on contrast agents. Most common contrast agents are microbubbles that are confined to vascular compartments and molecular targets because micrometer size particles cannot escape through endothelial barriers. Therefore, a desired ultrasound contrast agent should consist of nanometer scale particles that are capable of escaping from vasculature, penetrating into tissue, and then generating sufficient contrast once they reach the target site. We developed a novel contrast agent—plasmonic nanoparticles covered by azide compounds (AzNPs), capable of on-demand laser-induced gas generation via photolysis of azide functional groups. The 50 nm diameter AzNPs were small enough to penetrate the endothelial barrier. Upon laser activation, the AzNPs generated nitrogen gas which served as ultrasound contrast agent. The feasibility of the AzNPs to produce ultrasound contrast was tested within polyethylene tube under laminar flow. Upon laser irradiation, significant ultrasound signal enhancement was observed due to the N2 gas bubbles generated by the photolysis of AzNPs. These and other studies suggest that the AzNPs may enable the ultrasound diagnosis of various diseases that conventional microbubbles cannot detect. Furthermore, these optically absorbing particles can also be used for imaging and therapeutic applications of light and sound such as ultrasound-guided photoacoustic imaging.

Metastases, rather than primary tumors, are responsible for majority of deaths in cancer patients. We developed a non-invasive method to detect sentinel lymph node (SLN) metastasis using combined ultrasound and photoacoustic (US/PA) imaging with glycol-chitosan-coated gold nanoparticles (GC-AuNP). The spatio-temporal distribution of GC-AuNP is affected by presence of metastasis, which is detected by US/PA. GC-AuNPs (100 μl, 0.1 mg Au/ml) were injected peritumorally in breast tumor-bearing mice, and allowed to drain for 24 hours into the SLN. The SLN was located using b-mode ultrasound, and multi-wavelength PA was used to detect GC-AuNPs. Spectroscopic analysis allowed to isolate the signal from GC-AuNPs and quantify their accumulation in the SLN after cellular uptake and transport by immune cells. Results show that distribution of GC-AuNPs in the SLN was affected by presence of metastasis. The overall accumulation was also reduced, with more than 20% signal decrease compared to non-metastatic controls. Histological analysis confirmed that distribution of GC-AuNP-containing immune cells is reduced due to presence of metastatic cells. Our method successfully distinguishes metastatic from non-metastatic lymph nodes using biocompatible nanoparticles and could aid physicians in detection of micrometastasis thus guiding and avoiding unnecessary SLN biopsy.

Shear wave elasticity imaging (SWEI), an emerging acoustic technology for medical diagnostics, is based on remote generation of shear waves in tissue by radiation force in the focal region of an ultrasonic beam. In this study, the feasibility of Doppler ultrasonic technique to visualize the remotely induced shear waves was demonstrated. The generation of shear displacement in the focal region of a pulsed 1-MHz ultrasound beam with pulse duration of approximately about 2 ms and intensity levels on the order of 145 W/cm2, and consequent propagation of shear wave in tissue-mimicking and muscle tissue in vitro, were measured. The analysis of temporal behavior of shear displacement within the focal plane allowed estimation of shear wave velocities. The velocities were 4 and 7 m/s in hard phantom and tissue containing phantom, respectively. The measured shear displacements on the order of micrometers in gel-based phantoms are in reasonable agreement with theoretical estimates derived from an earlier developed model of shear wave generation by radiation force of focused ultrasound. The study revealed significant dependence of shear strain on the medium viscosity. The complex oscillatory character of shear strain relaxation in viscoelastic phantom and muscle tissue in vitro was observed.

To obtain high sensitivity, specificity, and signal/contrast-to-noise ratio imaging of disease, detection of biomarkers or biochemical and cellular processes is often needed. Typically, such information is gathered using imaging contrast agents. Unfortunately, microbubbles—most common ultrasound contrast agent, are too large to escape vascular compartments. Thus, contrast-enhanced molecular ultrasound imaging is often limited to molecular markers on the vascular endothelium and other intravascular targets. Recently, we introduced several high-resolution, high-sensitivity, depth-resolved ultrasound-based molecular imaging techniques augmented with nanometer scale imaging contrast agents capable of visualizing molecular signatures of cells in context of structural and functional properties of tissue. In this presentation, several examples of molecular ultrasound imaging based on synergistic combination of light and sound will be given. Specifically, clinical problems including detection and phenotyping of primary tumor, assessment of micrometastatic lesions in sentinel lymph node, and image-guided cancer cell therapy will be described. Design and synthesis of clinically relevant contrast nanoagents with properties desired for cellular/molecular ultrasound imaging will be discussed. Finally, the presentation will conclude with the analysis of how molecular ultrasound can change both fundamental medical science and the utility of clinical ultrasound imaging in diagnostic and therapeutic applications.

Acoustic radiation force on a scatterer in tissue depends on the compressibility and shear modulus of both the tissue and the scatterer. This force is related to the monopole and dipole scattering coefficients. The finite shear modulus of the tissue decreases the radiation force in comparison with the force exerted on the same scatterer surrounded by liquid. Shear moduli for soft tissue range from several kilopascals (breast, liver) to tens of kilopascals and higher for cornea, cartilage, and cancerous tissue. As reported previously, the radiation force on a bubble in tissue having 100 kPa shear modulus is 50% less than if the bubble is in water. This difference decreases for scatterers with finite shear moduli, examples of which are reported here. Additionally, displacement of a scatterer due to radiation force is inversely proportional to the shear modulus of the tissue, which permits measurement of the latter. Experiments demonstrating this technique are reviewed. In these experiments, the radiation force is applied to a gas microbubble produced by laser-induced optical breakdown, while displacement of the microbubble is measured by high-frequency ultrasound as a function of time. Results are reported for tissue-mimicking phantoms and animal crystalline lenses in vitro.

Microbubbles are already widely used for biomedical and clinical purposes, and are further being investigated for innovative medical and industrial applications. In most cases, the Rayleigh–Plesset equation for a gas bubble in liquid is used. In this paper we present a more general equation for gas bubble dynamics in viscoelastic media such as soft tissue. The equation, describing oscillations of a spherical bubble in an isotropic, incompressible medium, contains an integral over an elastic energy density function that depends on two invariants of the strain tensor. For moderate bubble oscillations one may use Landau’s cubic expansion of energy density, which contains five elastic moduli—shear and bulk moduli corresponding to quadratic terms, and three moduli (A, B, and C) corresponding to cubic terms. With energy density expressed through invariants of the strain tensor in spherical coordinates, the total elastic energy for an incompressible medium becomes an explicit function of bubble radius and contains only two of the five moduli, the shear modulus and modulus A. Compared to the Rayleigh–Plesset equation, the resulting equation has additional terms associated with shear force and elastic nonlinearity. Further modifications of this equation account for weak compressibility, viscosity, surface tension, radiation loss, and elastic shells.

According to the most widely accepted theory of presbyopia, the age-related loss of accommodation is attributed to Young's modulus changes in the lens. Previously, we have developed an approach to measure the mechanical properties of viscoelastic medium using microbubbles and acoustic radiation force. In this study, we tested this technique to assess the mechanical properties of bovine lenses ex-vivo. An impulsive acoustic radiation force was applied to laser-induced microbubbles created with nanosecond laser pulses at different locations in the lens. An acoustic radiation force with typical duration less than 150 μs was generated by a 3.5 MHz transducer. For accurate spatio-temporal measurements of the microbubble's displacement, a custom-made ultrasound system consisting of two 25 MHz transducers was built. The first transducer emitted a train of pulses and the other transducer received the train of echoes reflected from the microbubble. The developed system was operating at 400 kHz pulse repetition fre...

Macrophages and lipid are two key components in the development and characterization of atherosclerotic plaques. Imaging the activity and distribution of macrophages and lipid in the vessel wall would help identify the vulnerable plaques. In this study, spectroscopic intravascular photoacoustic (sIVPA) imaging was used to detect phagocytically active macrophages and deposits of lipid simultaneously. Polyethylene glycol coated spherical gold nanoparticles (Au NPs) were intravenously injected in vivo into an atherosclerotic rabbit allowing phagocytically active macrophages within the plaques to be labeled with Au NPs. Atherosclerotic aorta was harvested and scanned with a bench‐top sIVPA imaging system. Au NP‐labeled macrophages were detected in the 710–770 nm wavelength range because of the plasmon resonance coupling effect. Lipid was imaged in 1210–1230 nm wavelength range based on the characteristic optical absorption spectrum of fatty acids. An image processing method was developed to identify Au‐NP‐lab...

A morphological, functional, and molecular imaging technique capable of visualizing biochemical, pharmacological, and other processes in vivo and repetitively during various stages of tumor progression and cancer treatment is desired for many biomedical, preclinical, and clinical applications. Over the past several years, we have been developing an integrated imaging approach—ultrasound‐guided photoacoustic imaging. Ultrasound is used to visualize anatomical structures and photoacoustics is used to provide functional information about tissue using spectral differences between absorption of oxygenated and deoxygenated hemoglobin and cellular activity using molecularly targeted plasmonic nanosensors. In this paper, the ability of ultrasound and nanoparticle‐mediated photoacoustic imaging to simultaneously obtain the anatomical and molecular map of tumor in vivo will be demonstrated. Furthermore, the role of ultrasound and photoacoustic imaging in therapy of cancer will be discussed. Finally, the development...

A unique contrast agent, termed photoacoustic nanodroplet (PAnD), has been developed for several biomedical and clinical applications. A PAnD consists of several plasmonic nanoparticles encapsulated within a nano‐sized perfluorocarbon droplet. PAnDs act as contrast agents for ultrasound and photoacoustic imaging through different mechanisms. Optical absorption by plasmonic nanoparticles triggers vaporization of the perfluorocarbon droplet, yielding two effects: first, strong photoacoustic transients are generated, and second, a gas microbubble is formed. Furthermore, subsequent laser pulses interacting with expelled plasmonic nanoparticles result in photoacoustic transients due to thermoelastic expansion of tissue. To demonstrate these contrast enhancement mechanisms, a homogenously laden polyacrylamide phantom (108 PAnD/ml) underwent pulsed laser irradiation (5 mJ/cm2, 10 Hz PRF, 5–7 ns pulse duration), while photoacoustic and ultrasound images were captured with a 40 MHz linear array transducer. Photoac...

Measurements of mechanical properties of tissue can be used in many applications ranging from disease diagnosis to treatment planning and monitoring. In this paper, we present a technique to measure mechanical properties of the tissue undergoing laser surgery during which the cavitation bubble is introduced. Utilizing the laser‐induced microbubble, the mechanical properties of the tissue were assessed by measuring displacement and oscillations of the bubble under applied acoustic radiation force. The bubbles were produced using a nanosecond pulsed laser operating at 532 nm. Using the lens (0.92 numerical aperture, 11.5‐mm focal length), the laser beam was focused inside the gelatin to create microbubbles ranging from 80‐ to 250‐cm diameter. The bubbles were then interrogated using acoustic pulses of different durations and amplitudes. First, the size of each laser‐induced microbubble was estimated by measuring the laser‐produced shock wave and pulse‐echo ultrasound signals. Then, a 1.5‐MHz transducer gene...

An accurate assessment of mechanical properties of tissue is desired in many applications ranging from biomedical research to medical diagnostics and surgery. Currently, several methods to assess shear elasticity of tissue are available while the techniques to measure tissue viscosity are not yet fully developed. However, shear viscosity is an indicative functional parameter of soft tissue. In addition, the local changes of shear viscosity can affect the accuracy of shear elasticity assessment. Therefore, in this study, an acoustic radiation force approach to assess both shear elasticity and viscosity of tissue is developed. Such approach is based on monitoring of the spatio‐temporal displacement of an ultrasound inhomogeneity in response to applied impulsive radiation force. In experiments, a 2‐mm‐diameter solid sphere was embedded into the gel phantoms with varying shear elasticity and viscosity. The sphere was then perturbed using acoustic radiation force produced by a single‐element focused ultrasound...

Photoacoustics is a wonder as the lightning and thunder once were. Indeed, the absorbed short laser light pulses can generate sound. Utilizing this photoacoustic effect, researchers are now combining photonics with ultrasound technologies, nanoscience, and molecular biology to develop new approaches in molecular imaging and therapy. In photoacoustic imaging, the tissue is irradiated with non‐ionizing laser pulses. The deep‐penetrating photons are preferentially absorbed within the tissue and, through thermoelastic expansion, the photoacoustic transients are generated. Using acoustic sensors, these photoacoustic waves are then detected at the tissue surface to assess the strength and distribution of optically induced internal sound sources. Photoacoustic imaging can be seamlessly fused with ultrasound imaging, taking full advantage of the many synergistic features of these systems. Furthermore, using bioconjugated nanoparticles with high optical absorption, events at the molecular and cellular levels can b...

Catheter‐based intravascular ultrasound (IVUS) is one of the imaging tools for the clinical evaluation of atherosclerosis. However, histopathological information obtained with IVUS imaging is limited. We developed a combined intravascular photoacoustic (IVPA) and IVUS imaging to characterize atherosclerotic plaques. The amplitude, spectral, and temporal characteristics of the photoacoustic signal are primarily determined by optical absorption properties of different types of tissues and can be used to differentiate the lipid, blood, fibrous, and fibro‐cellular components of an inflammatory lesion. Furthermore, using bioconjugated contrast agents such as metal nanoconstructs, molecular IVPA imaging is possible since different molecules are overexpressed during various stages of atherosclerosis. Imaging experiments were performed using clinical IVUS imaging catheters interfaced with a pulsed laser system. The performance of the IVPA/IVUS imaging was assessed using vessel‐mimicking phantoms. To detect the li...

Laser therapy is an alternative cancer treatment approach when traditional surgery cannot be used to remove small, poorly defined lesions embedded within vital organs. For example, in photothermal laser therapy, a localized temperature increase to cause tumor necrosis is achieved by using a continuous wave laser and molecular targeted and optically tuned photoabsorbers. However, for laser therapy to be successful, it is necessary to identify the presence of photoabsorbers in the tumor prior to therapy, to monitor the spatio‐temporal temperature changes during therapy, and, finally, to identify thermal/mechanical damage and tumor destruction after therapy. To guide laser therapy, we developed a combined ultrasound/photoacoustic imaging system and bioconjugated nanoabsorbers. In our approach, ultrasound imaging is utilized to identify the anatomy of the tumor while photoacoustic imaging is used to ensure the presence of targeted nanoparticles before therapy. Both ultrasound and photoacoustic imaging techniq...

Lasers are used in many biomedical and clinical applications, ranging from diagnosis to therapy. In eye microsurgery, for example, a pulsed laser beam produces a localized surgical effect through the formation of a cavitation microbubble as a result of tissue photodisruption occurring in the focal zone. However, to insure successful presurgical planning, surgical procedure, and postoperative stages of pathology treatment, the mechanical properties of the tissue must be analyzed before selective laser intervention. Since microbubbles are already produced during laser surgery, we have developed an integrated approach utilizing these laser‐induced microbubbles as reporters of tissue viscoelasticity. Specifically, we have derived a general model of gas bubble dynamics in viscoelastic media to describe both translation and oscillations of the microbubble exposed to the acoustic radiation force. Furthermore, an ultrasound method based on temporal measurement of passive acoustic emission from cavity during laser...

Measurement of small‐bubble dynamics has been proposed for the remote evaluation of tissue elasticity [Erpelding et al., Proc. IEEE Ultrasonics Symp., 554–557, 2003]. For example, a microbubble can be produced within the cornea during femtosecond laser surgery and its response to a pulsed ultrasonic radiation force can be measured. The bubble’s translation, deformation, and oscillation can be directly related to the mechanical properties of surrounding tissue information that is required for optimization of the surgical procedure. In the work reported here, a model was developed to predict the translation and deformation of an initially spherical bubble in a soft viscoelastic medium as induced by radiation pressure. The extent of bubble translation and deformation is dictated by the elastic stress and viscous forces that oppose the radiation pressure. Numerical simulations predict static, periodic, and transient translation of the bubble in response to continuous, periodic, and pulsed waveforms, respectively. The model also predicts the deformation of the bubble. The results indicate increased deformations with increased bubble translations. Overall, the model can be used to determine the local shear modulus and viscosity of the medium based on measurements of gas‐bubble displacement. [Work supported by ARL:UT IR&D.]

The effective management of cancer requires early yet reliable detection, localization, and diagnosis. Therefore, there is a definite and urgent clinical need for an imaging technique that is widely available, is simple to perform, is safe, and that can detect and adequately diagnose cancer. In this paper we present a hybrid imaging technology based on fusion of complementary imaging modalities ultrasound, optoacoustics, and elastography to take full advantage of the many synergistic features of these modalities and thus to significantly improve sensitivity and specificity of cancer imaging. To evaluate our approach, numerical and experimental studies were performed using heterogeneous phantoms where ultrasonic, optical, and viscoelastic properties of the materials were chosen to closely mimic soft tissue. The results of this study suggest that combined ultrasound‐based imaging is possible and can provide more accurate, reliable and earlier detection and diagnosis of tissue pathology. In addition, monitoring of cancer treatment and guidance of tissue biopsy are possible with combined imaging system. Practical and experimental aspects of combined imaging will be discussed with emphasis on data capture and signal/image processing algorithms. The paper will conclude with a discussion of the advantages, limitations, and potential clinical applications of the combined imaging technique.

Nonlinear oscillations of microbubbles are being investigated extensively for innovative medical and industrial applications. The Rayleigh–Plesset equation successfully describes the dynamics of gas bubbles in liquids and viscoelastic media. To model bubble dynamics in weakly compressible elastic media such as soft tissue, effects of shear stress should be taken into account. These effects were included recently by the authors in a model for bubble oscillations that differs from the Rayleigh–Plesset equation by a single term associated with nonlinear elasticity of the medium surrounding the bubble. Here, we augment this dynamical equation to account for acoustic radiation and thus describe nonlinear bubble oscillations in slightly compressible elastic media. The bubble is assumed to be spherical and the surrounding elastic medium to be isotropic. The analysis is performed in Eulerian coordinates, and attention is devoted to terms associated with radiation loss. Our analysis builds on results obtained by Prosperetti and Lezzi [J. Fluid Mech. 168, 457 (1986)] for a bubble in liquid. The stress tensor for the elastic medium is expressed through invariants of the Green deformation tensor in spherical coordinates. Explicit expressions for components of the stress tensor are obtained using Mooney’s potential function. [Work supported by IR&D at ARL:UT.]