Tri-modality cavitation mapping in shock wave lithotripsy.

Shock wave lithotripsy (SWL) has been widely used for non-invasive treatment of kidney stones. Cavitation plays an important role in stone fragmentation, yet it may also contribute to renal injury during SWL. It is therefore crucial to determine the spatiotemporal distributions of cavitation activities to maximize stone fragmentation while minimizing tissue injury. Traditional cavitation detection methods include high-speed optical imaging, active cavitation mapping (ACM), and passive cavitation mapping (PCM). While each of the three methods provides unique information about the dynamics of the bubbles, PCM has most practical applications in biological tissues. To image the dynamics of cavitation bubble collapse, we previously developed a sliding-window PCM (SW-PCM) method to identify each bubble collapse with high temporal and spatial resolution. In this work, to further validate and optimize the SW-PCM method, we have developed tri-modality cavitation imaging that includes three-dimensional high-speed optical imaging, ACM, and PCM seamlessly integrated in a single system. Using the tri-modality system, we imaged and analyzed laser-induced single cavitation bubbles in both free field and constricted space and shock wave-induced cavitation clusters. Collectively, our results have demonstrated the high reliability and spatial-temporal accuracy of the SW-PCM approach, which paves the way for the future in vivo applications on large animals and humans in SWL.

Variations of stress field and stone fracture produced at different lateral locations in a shockwave lithotripter field.

During clinical procedures, the lithotripter shock wave (LSW) that is incident on the stone and resultant stress field is often asymmetric due to the respiratory motion of the patient. The variations of the LSW-stone interaction and associated fracture pattern were investigated by photoelastic imaging, phantom experiments, and three-dimensional fluid-solid interaction modeling at different lateral locations in a lithotripter field. In contrast to a T-shaped fracture pattern often observed in the posterior region of the disk-shaped stone under symmetric loading, the fracture pattern gradually transitioned to a tilted L-shape under asymmetric loading conditions. Moreover, the model simulations revealed the generation of surface acoustic waves (SAWs), i.e., a leaky Rayleigh wave on the anterior boundary and Scholte wave on the posterior boundary of the stone. The propagation of SAWs on the stone boundary is accompanied by a progressive transition of the LSW reflection pattern from regular to von Neumann and to weak von Neumann reflection near the glancing incidence and, concomitantly, the development and growth of a Mach stem, swirling around the stone boundary. The maximum tensile stress and stress integral were produced by SAWs on the stone boundary under asymmetric loading conditions, which drove the initiation and extension of surface cracks into the bulk of the stone that is confirmed by micro-computed tomography analysis.

Improving the lens design and performance of a contemporary electromagnetic shock wave lithotripter.

The efficiency of shock wave lithotripsy (SWL), a noninvasive first-line therapy for millions of nephrolithiasis patients, has not improved substantially in the past two decades, especially in regard to stone clearance. Here, we report a new acoustic lens design for a contemporary electromagnetic (EM) shock wave lithotripter, based on recently acquired knowledge of the key lithotripter field characteristics that correlate with efficient and safe SWL. The new lens design addresses concomitantly three fundamental drawbacks in EM lithotripters, namely, narrow focal width, nonidealized pulse profile, and significant misalignment in acoustic focus and cavitation activities with the target stone at high output settings. Key design features and performance of the new lens were evaluated using model calculations and experimental measurements against the original lens under comparable acoustic pulse energy (E+) of 40 mJ. The -6-dB focal width of the new lens was enhanced from 7.4 to 11 mm at this energy level, and peak pressure (41 MPa) and maximum cavitation activity were both realigned to be within 5 mm of the lithotripter focus. Stone comminution produced by the new lens was either statistically improved or similar to that of the original lens under various in vitro test conditions and was significantly improved in vivo in a swine model (89% vs. 54%, P = 0.01), and tissue injury was minimal using a clinical treatment protocol. The general principle and associated techniques described in this work can be applied to design improvement of all EM lithotripters.

Assessment of a modified acoustic lens for electromagnetic shock wave lithotripters in a swine model.

PURPOSE: The acoustic lens of the Modularis electromagnetic shock wave lithotripter (Siemens, Malvern, Pennsylvania) was modified to produce a pressure waveform and focal zone more closely resembling that of the original HM3 device (Dornier Medtech, Wessling, Germany). We assessed the newly designed acoustic lens in vivo in an animal model. MATERIALS AND METHODS: Stone fragmentation and tissue injury produced by the original and modified lenses of the Modularis lithotripter were evaluated in a swine model under equivalent acoustic pulse energy (about 45 mJ) at 1 Hz pulse repetition frequency. Stone fragmentation was determined by the weight percent of stone fragments less than 2 mm. To assess tissue injury, shock wave treated kidneys were perfused, dehydrated, cast in paraffin wax and sectioned. Digital images were captured every 120 µm and processed to determine functional renal volume damage. RESULTS: After 500 shocks, the mean ± SD stone fragmentation efficiency produced by the original and modified lenses was 48% ± 12% and 52% ± 17%, respectively (p = 0.60). However, after 2,000 shocks, the modified lens showed significantly improved stone fragmentation compared to the original lens (mean 86% ± 10% vs 72% ± 12%, p = 0.02). Tissue injury caused by the original and modified lenses was minimal at a mean of 0.57% ± 0.44% and 0.25% ± 0.25%, respectively (p = 0.27). CONCLUSIONS: With lens modification the Modularis lithotripter demonstrates significantly improved stone fragmentation with minimal tissue injury at a clinically relevant acoustic pulse energy. This new lens design could potentially be retrofitted to existing lithotripters, improving the effectiveness of electromagnetic lithotripters.

Experimentally validated multiphysics computational model of focusing and shock wave formation in an electromagnetic lithotripter.

A multiphysics computational model of the focusing of an acoustic pulse and subsequent shock wave formation that occurs during extracorporeal shock wave lithotripsy is presented. In the electromagnetic lithotripter modeled in this work the focusing is achieved via a polystyrene acoustic lens. The transition of the acoustic pulse through the solid lens is modeled by the linear elasticity equations and the subsequent shock wave formation in water is modeled by the Euler equations with a Tait equation of state. Both sets of equations are solved simultaneously in subsets of a single computational domain within the BEARCLAW framework which uses a finite-volume Riemann solver approach. This model is first validated against experimental measurements with a standard (or original) lens design. The model is then used to successfully predict the effects of a lens modification in the form of an annular ring cut. A second model which includes a kidney stone simulant in the domain is also presented. Within the stone the linear elasticity equations incorporate a simple damage model.

A Multi-Spark Electrohydraulic Shock Wave Generator with Adjustable Pressure Field Distribution and Beam Steering Capability.

Background and objective: All clinical shock wave lithotripters produce an axisymmetric acoustic field without accounting for the anatomic features of the kidney or respiratory motion of the patient. This work presents a steerable and adjustable focusing electrohydraulic (SAFE) shock wave generator design with variable beam size and shape. Materials and Methods: 90 electrohydraulic transducers are mounted concentrically on a spherical basin with adjustable connection to individual transducers. Each transducer consists of 45 3D-printed titanium microelectrodes embedded in epoxy with a tip diameter of 0.3 mm. All the transducers are arranged in 5 concentric rings and sub-divided into 6 sectors. Results: By changing the connections of individual transducers, the focused pressure field produced by the transducer array can be either axisymmetric with a -6 dB focal width of 14.8 mm in diameter, or non-axisymmetric with a long axis of 22.7 mm and a short axis of 15.1 mm. The elongated beam produces a peak positive pressure of 33.7±4.1 MPa and comminution efficiency of 42.2±3.5%, compared to 36.2±0.7 MPa and 28.6±6.1% for axisymmetric beam after 150 pulses at 20 kV. Conclusions: We have demonstrated that the SAFE shock wave generator can produce an elongated non-axisymmetric pressure field with higher stone comminution efficiency. The SAFE shock wave generator may provide a flexible and versatile design to achieve accurate, stable, and safe lithotripsy for kidney stone treatment.

Shock waves generated by toroidal bubble collapse are imperative for kidney stone dusting during Holmium:YAG laser lithotripsy.

Holmium:yttrium-aluminum-garnet (Ho:YAG) laser lithotripsy (LL) has been the treatment of choice for kidney stone disease for more than two decades, yet the mechanisms of action are not completely clear. Besides photothermal ablation, recent evidence suggests that cavitation bubble collapse is pivotal in kidney stone dusting when the Ho:YAG laser operates at low pulse energy (Ep) and high frequency (F). In this work, we perform a comprehensive series of experiments and model-based simulations to dissect the complex physical processes in LL. Under clinically relevant dusting settings (Ep = 0.2 J, F = 20 Hz), our results suggest that majority of the irradiated laser energy (>90 %) is dissipated by heat generation in the fluid surrounding the fiber tip and the irradiated stone surface, while only about 1 % may be consumed for photothermal ablation, and less than 0.7 % is converted into the potential energy at the maximum bubble expansion. We reveal that photothermal ablation is confined locally to the laser irradiation spot, whereas cavitation erosion is most pronounced at a fiber tip-stone surface distance about 0.5 mm where multi foci ring-like damage outside the thermal ablation zone is observed. The cavitation erosion is caused by the progressively intensified collapse of jet-induced toroidal bubble near the stone surface (<100 µm), as a result of Raleigh-Taylor and Richtmyer-Meshkov instabilities. The ensuing shock wave-stone interaction and resultant leaky Rayleigh waves on the stone surface may lead to dynamic fatigue and superficial material removal under repeated bombardments of toroidal bubble collapses during dusting procedures in LL.

Dissimilar cavitation dynamics and damage patterns produced by parallel fiber alignment to the stone surface in holmium:yttrium aluminum garnet laser lithotripsy.

Recent studies indicate that cavitation may play a vital role in laser lithotripsy. However, the underlying bubble dynamics and associated damage mechanisms are largely unknown. In this study, we use ultra-high-speed shadowgraph imaging, hydrophone measurements, three-dimensional passive cavitation mapping (3D-PCM), and phantom test to investigate the transient dynamics of vapor bubbles induced by a holmium:yttrium aluminum garnet laser and their correlation with solid damage. We vary the standoff distance (SD) between the fiber tip and solid boundary under parallel fiber alignment and observe several distinctive features in bubble dynamics. First, long pulsed laser irradiation and solid boundary interaction create an elongated "pear-shaped" bubble that collapses asymmetrically and forms multiple jets in sequence. Second, unlike nanosecond laser-induced cavitation bubbles, jet impact on solid boundary generates negligible pressure transients and causes no direct damage. A non-circular toroidal bubble forms, particularly following the primary and secondary bubble collapses at SD = 1.0 and 3.0 mm, respectively. We observe three intensified bubble collapses with strong shock wave emissions: the intensified bubble collapse by shock wave, the ensuing reflected shock wave from the solid boundary, and self-intensified collapse of an inverted "triangle-shaped" or "horseshoe-shaped" bubble. Third, high-speed shadowgraph imaging and 3D-PCM confirm that the shock origins from the distinctive bubble collapse form either two discrete spots or a "smiling-face" shape. The spatial collapse pattern is consistent with the similar BegoStone surface damage, suggesting that the shockwave emissions during the intensified asymmetric collapse of the pear-shaped bubble are decisive for the solid damage.

Cavitation Plays a Vital Role in Stone Dusting During Short Pulse Holmium:YAG Laser Lithotripsy.

Objective: To investigate the mechanism of stone dusting in Holmium (Ho): YAG laser lithotripsy (LL). Materials and Methods: Cylindrical BegoStone samples (6 × 6 mm, H × D) were treated in water using a clinical Ho:YAG laser lithotripter in dusting mode (0.2-0.4 J with 70-78 µs in pulse duration, 20 Hz) at various fiber tip to stone standoff distances (SD = 0, 0.5, and 1 mm). Stone damage craters were quantified by optical coherence tomography and bubble dynamics were captured by high-speed video imaging. To differentiate the contribution of cavitation vs thermal ablation to stone damage, three additional experiments were performed. First, presoaked wet stones were treated in air to assess stone damage without cavitation. Second, the laser fiber was advanced at various offset distances (OSD = 0.25, 1, 2, 3, and 10 mm) from the tip of a flexible ureteroscope to alter the dynamics of bubble collapse. Third, stones were treated with parallel fiber to minimize photothermal damage while isolating the contribution of cavitation to stone damage. Results: Treatment in water resulted in 2.5- to 90-fold increase in stone damage compared with those produced in air where thermal ablation dominates. With the fiber tip placed at OSD = 0.25 mm, the collapse of the bubble was distracted away from the stone surface by the ureteroscope tip, leading to significantly reduced stone damage compared with treatment without the scope or with scope at large OSD of 3-10 mm. The average crater volume produced by parallel fiber orientation at 0.2 J after 100 pulses, where cavitation is the dominant mechanism of stone damage, was comparable with those produced by using perpendicular fiber orientation within SD = 0.25-1 mm. Conclusion: Cavitation plays a dominant role over photothermal ablation in stone dusting during short pulse Ho:YAG LL when 10 or more pulses are delivered to the same location.

Dynamics of tandem bubble interaction in a microfluidic channel.

The dynamics of tandem bubble interaction in a microfluidic channel (800 × 21 µm, W × H) have been investigated using high-speed photography, with resultant fluid motion characterized by particle imaging velocimetry. A single or tandem bubble is produced reliably via laser absorption by micron-sized gold dots (6 µm in diameter with 40 µm in separation distance) coated on a glass surface of the microfluidic channel. Using two pulsed Nd:YAG lasers at λ = 1064 nm and ∼10 µJ/pulse, the dynamics of tandem bubble interaction (individual maximum bubble diameter of 50 µm with a corresponding collapse time of 5.7 µs) are examined at different phase delays. In close proximity (i.e., interbubble distance = 40 µm or γ = 0.8), the tandem bubbles interact strongly with each other, leading to asymmetric deformation of the bubble walls and jet formation, as well as the production of two pairs of vortices in the surrounding fluid rotating in opposite directions. The direction and speed of the jet (up to 95 m/s), as well as the orientation and strength of the vortices can be varied by adjusting the phase delay.

Time-Resolved Passive Cavitation Mapping Using the Transient Angular Spectrum Approach.

Passive cavitation mapping (PCM), which generates images using bubble acoustic emission signals, has been increasingly used for monitoring and guiding focused ultrasound surgery (FUS). PCM can be used as an adjunct to magnetic resonance imaging to provide crucial information on the safety and efficacy of FUS. The most widely used algorithm for PCM is delay-and-sum (DAS). One of the major limitations of DAS is its suboptimal computational efficiency. Although frequency-domain DAS can partially resolve this issue, such an algorithm is not suitable for imaging the evolution of bubble activity in real time and for cases in which cavitation events occur asynchronously. This study investigates a transient angular spectrum (AS) approach for PCM. The working principle of this approach is to backpropagate the received signal to the domain of interest and reconstruct the spatial-temporal wavefield encoded with the bubble location and collapse time. The transient AS approach is validated using an in silico model and water bath experiments. It is found that the transient AS approach yields similar results to DAS, but it is one order of magnitude faster. The results obtained by this study suggest that the transient AS approach is promising for fast and accurate PCM.

Internal-Illumination Photoacoustic Tomography Enhanced by a Graded-Scattering Fiber Diffuser.

The penetration depth of photoacoustic imaging in biological tissues has been fundamentally limited by the strong optical attenuation when light is delivered externally through the tissue surface. To address this issue, we previously reported internal-illumination photoacoustic imaging using a customized radial-emission optical fiber diffuser, which, however, has complex fabrication, high cost, and non-uniform light emission. To overcome these shortcomings, we have developed a new type of low-cost fiber diffusers based on a graded-scattering method in which the optical scattering of the fiber diffuser is gradually increased as the light travels. The graded scattering can compensate for the optical attenuation and provide relatively uniform light emission along the diffuser. We performed Monte Carlo numerical simulations to optimize several key design parameters, including the number of scattering segments, scattering anisotropy factor, divergence angle of the optical fiber, and reflective index of the surrounding medium. These optimized parameters collectively result in uniform light emission along the fiber diffuser and can be flexibly adjusted to accommodate different applications. We fabricated and characterized the prototype fiber diffuser made of agarose gel and intralipid. Equipped with the new fiber diffuser, we performed thorough proof-of-concept studies on ex vivo tissue phantoms and an in vivo swine model to demonstrate the deep-imaging capability (~10 cm achieved ex vivo) of photoacoustic tomography. We believe that the internal light delivery via the optimized fiber diffuser is an effective strategy to image deep targets (e.g., kidney) in large animals or humans.

The Role of Cavitation in Energy Delivery and Stone Damage During Laser Lithotripsy.

Purpose: Although cavitation during laser lithotripsy (LL) contributes to the Moses effect, the impact of cavitation on stone damage is less clear. Using different laser settings, we investigate the role of cavitation bubbles in energy delivery and stone damage. Materials and Methods: The role of cavitation in laser energy delivery was characterized by using photodetector measurements synced with high-speed imaging for laser pulses of varying durations. BegoStone samples were treated with the laser fiber oriented perpendicularly in contact with the stone in water or in air to assess the impact of cavitation on crater formation. Crater volume and geometry were quantified by using optical coherence tomography. Further, the role of cavitation in stone damage was elucidated by treatment in water with the fiber oriented parallel to the stone surface and by photoelastic imaging. Results: Longer pulse durations resulted in higher energy delivery but smaller craters. Stones treated in water resulted in greater volume, wider yet shallower craters compared with those treated in air. Stones treated with the parallel fiber showed crater formation after 15 pulses, confirmed by high-speed imaging of the bubble collapse with the resultant stress field captured by photoelastic imaging. Conclusions: Despite improved energy delivery, the longer pulse mode produced smaller crater volume, suggesting additional processes secondary to photothermal ablation are involved in stone damage. Our critical observations of the difference in stone damage treated in water vs in air, combined with the crater formation by parallel fiber, suggest that cavitation is a contributor to stone damage during LL.

A simple method for fabricating artificial kidney stones of different physical properties.

A simple method for preparing artificial kidney stones with varying physical properties is described. BegoStone was prepared with a powder-to-water ratio ranging from 15:3 to 15:6. The acoustic properties of the phantoms were characterized using an ultrasound transmission technique, from which the corresponding mechanical properties were calculated based on elastic wave theory. The measured parameters for BegoStone phantoms of different water contents are: longitudinal wave speed (3,148-4,159 m/s), transverse wave speed (1,813-2,319 m/s), density (1,563-1,995 kg/m(3)), longitudinal acoustic impedance (4.92-8.30 kg/m(2) s), transverse acoustic impedance (2.83-4.63 kg/m(2) s), Young's modulus (12.9-27.4 GPa), bulk modulus (8.6-20.2 GPa), and shear modulus (5.1-10.7 GPa), which cover the range of corresponding properties reported in natural kidney stones. In addition, diametral compression tests were carried out to determine tensile failure strength of the stone phantoms. BegoStone phantoms with varying water content at preparation have tensile failure strength from 6.9 to 16.3 MPa when tested dry and 3.2 to 7.1 MPa when tested in water-soaked condition. Overall, it is demonstrated that this new BegoStone preparation method can be used to fabricate artificial stones with physical properties matched with those of natural kidney stones of various chemical compositions.

Effect of lithotripter focal width on stone comminution in shock wave lithotripsy.

Using a reflector insert, the original HM-3 lithotripter field at 20 kV was altered significantly with the peak positive pressure (p(+)) in the focal plane increased from 49 to 87 MPa while the -6 dB focal width decreased concomitantly from 11 to 4 mm. Using the original reflector, p(+) of 33 MPa with a -6 dB focal width of 18 mm were measured in a pre-focal plane 15-mm proximal to the lithotripter focus. However, the acoustic pulse energy delivered to a 28-mm diameter area around the lithotripter axis was comparable ( approximately 120 mJ). For all three exposure conditions, similar stone comminution ( approximately 70%) was produced in a mesh holder of 15 mm after 250 shocks. In contrast, stone comminution produced by the modified reflector either in a 15-mm finger cot (45%) or in a 30-mm membrane holder (14%) was significantly reduced from the corresponding values (56% and 26%) produced by the original reflector (no statistically significant differences were observed between the focal and pre-focal planes). These observations suggest that a low-pressure/broad focal width lithotripter field will produce better stone comminution than its counterpart with high-pressure/narrow focal width under clinically relevant in vitro comminution conditions.

Thermal Memory Based Photoacoustic Imaging of Temperature.

Temperature mapping is essential in many biomedical studies and interventions to precisely control the tissue's thermal conditions for optimal treatment efficiency and minimal side effects. Based on the Grüneisen parameter's temperature dependence, photoacoustic (PA) imaging can provide relative temperature measurement, but it has been traditionally challenging to measure absolute temperatures without knowing the baseline temperature, especially in deep tissues with unknown optical and acoustic properties. Here, we report a new thermal-energy-memory-based photoacoustic thermometry (TEMPT). By illuminating the tissue with a burst of nanosecond laser pulses, TEMPT exploits the temperature dependence of the thermal energy lingering, which is probed by the corresponding PA signals acquired within the thermal confinement. A self-normalized ratiometric measurement cancels out temperature-irrelevant quantities and estimates the Grüneisen parameter. The temperature can then be evaluated, given the tissue's temperature-dependent Grüneisen parameter, mass density, and specific heat capacity. Unlike the conventional PA thermometry, TEMPT does not require the knowledge of tissue's baseline temperature, nor the optical properties. We have developed a mathematical model to describe the temperature dependence in TEMPT. We have demonstrated the feasibility of the temperature evaluation on tissue phantoms at 1.5 cm depth within a clinically relevant temperature range. Finally, as proof-of-concept, we applied TEMPT for temperature mapping during focused ultrasound treatment in mice in vivo at 2 mm depth. As a generic temperature mapping method, TEMPT is expected to find applications in thermotherapy of cancers on small animal models.

Focusing of shock waves induced by optical breakdown in water.

The focusing of laser-generated shock waves by a truncated ellipsoidal reflector was experimentally and numerically investigated. Pressure waveform and distribution around the first (F(1)) and second foci (F(2)) of the ellipsoidal reflector were measured. A neodymium doped yttrium aluminum garnet laser of 1046 nm wavelength and 5 ns pulse duration was used to create an optical breakdown at F(1), which generates a spherically diverging shock wave with a peak pressure of 2.1-5.9 MPa at 1.1 mm stand-off distance and a pulse width at half maximum of 36-65 ns. Upon reflection, a converging shock wave is produced which, upon arriving at F(2), has a leading compressive wave with a peak pressure of 26 MPa and a zero-crossing pulse duration of 0.1 mus, followed by a trailing tensile wave of -3.3 MPa peak pressure and 0.2 mus pulse duration. The -6 dB beam size of the focused shock wave field is 1.6 x 0.2 mm(2) along and transverse to the shock wave propagation direction. Formation of elongated plasmas at high laser energy levels limits the increase in the peak pressure at F(2). General features in the waveform profile of the converging shock wave are in qualitative agreement with numerical simulations based on the Hamilton model.

An experimentally-calibrated damage mechanics model for stone fracture in shock wave lithotripsy.

A damage model suggested by the Tuler-Butcher concept of dynamic accumulation of microscopic defects is obtained from experimental data on microcrack formation in synthetic kidney stones. Experimental data on appearance of microcracks is extracted from micro-computed tomography images of BegoStone simulants obtained after subjecting the stone to successive pulses produced by an electromagnetic shock-wave lithotripter source. Image processing of the data is used to infer statistical distributions of crack length and width in representative transversal cross-sections of a cylindrical stone. A high-resolution finite volume computational model, capable of accurately modeling internal reflections due to local changes in material properties produced by material damage is used to simulate the accumulation of damage due to successive shocks. Comparison of statistical distributions of microcrack formation in computation and experiment allows calibration of the damage model. The model is subsequently used to compute fracture of a different aspect-ratio cylindrical stone predicting concurrent formation of two main fracture areas as observed experimentally.

Investigation of HIFU-induced anti-tumor immunity in a murine tumor model.

BACKGROUND: High intensity focused ultrasound (HIFU) is an emerging non-invasive treatment modality for localized treatment of cancers. While current clinical strategies employ HIFU exclusively for thermal ablation of the target sites, biological responses associated with both thermal and mechanical damage from focused ultrasound have not been thoroughly investigated. In particular, endogenous danger signals from HIFU-damaged tumor cells may trigger the activation of dendritic cells. This response may play a critical role in a HIFU-elicited anti-tumor immune response which can be harnessed for more effective treatment. METHODS: Mice bearing MC-38 colon adenocarcinoma tumors were treated with thermal and mechanical HIFU exposure settings in order to independently observe HIFU-induced effects on the host's immunological response. In vivo dendritic cell activity was assessed along with the host's response to challenge tumor growth. RESULTS: Thermal and mechanical HIFU were found to increase CD11c+ cells 3.1-fold and 4-fold, respectively, as compared to 1.5-fold observed for DC injection alone. In addition, thermal and mechanical HIFU increased CFSE+ DC accumulation in draining lymph nodes 5-fold and 10-fold, respectively. Moreover, focused ultrasound treatments not only caused a reduction in the growth of primary tumors, with tumor volume decreasing by 85% for thermal HIFU and 43% for mechanical HIFU, but they also provided protection against subcutaneous tumor re-challenge. Further immunological assays confirmed an enhanced CTL activity and increased tumor-specific IFN-gamma-secreting cells in the mice treated by focused ultrasound, with cytotoxicity induced by mechanical HIFU reaching as high as 27% at a 10:1 effector:target ratio. CONCLUSION: These studies present initial encouraging results confirming that focused ultrasound treatment can elicit a systemic anti-tumor immune response, and they suggest that this immunity is closely related to dendritic cell activation. Because DC activation was more pronounced when tumor cells were mechanically lysed by focused ultrasound treatment, mechanical HIFU in particular may be employed as a potential strategy in combination with subsequent thermal ablations for increasing the efficacy of HIFU cancer treatment by enhancing the host's anti-tumor immunity.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)-Cell Interaction and the Resultant Bioeffects at the Single-cell Level.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.