Acoustic radiation force induced accumulation and dynamics of microbubbles on compliant surfaces.

Ultrasound stimulated microbubbles have been shown to be capable of breaking up blood clots through micro-scale interactions occurring near the clot surface. However, only a small fraction of bubbles circulating in the bloodstream will be in close proximity to such boundaries, where they must be to elicit therapeutic effects. Here, the accumulation and subsequent behavior of microbubbles displaced from an overlying flow channel to a boundary under radiation forces were examined. Experimental data were acquired using a novel high speed microscopy configuration and simulations were conducted to provide insight into the accumulation process. There was broad agreement between experiments and simulations, both indicating that the size distribution and number of bubbles arriving at the boundary depended on channel flow rate, applied pressure, and bubble concentration. For example, higher flow rates and lower pressures favored the accumulation of larger bubbles relative to the native agent distribution. Moreover, bubble dynamics were dependent on the surface type, exhibiting rapid translation along agarose gel surfaces whereas on fibrin surfaces, they accumulated in localized regions inducing repetitive strain cycles. The results indicate that the process of bringing bubbles from within a vessel to a boundary is complex and should be an important consideration in the development of therapeutic applications such as sonothrombolysis.

The microscale evolution of the erosion front of blood clots exposed to ultrasound stimulated microbubbles.

Serial two-photon microscopy of blood clots with fluorescently tagged fibrin networks was conducted during microbubble-mediated sonothrombolysis to examine the microscale evolution of the resulting erosion front. The development of a complex zonal erosion pattern was observed, comprised of a cell depleted layer of fibrin network overlying intact clot which then underwent progressive recession. The fibrin zone architecture was dependent on exposure conditions with 0.1 MPa causing no erosion, 0.39 MPa resulting in homogenous structure, and combination 0.39/0.96 MPa pulses forming large-scale tunnels. High speed imaging and Coulter counter data indicated the fibrin zone formation process involves the ejection of intact erythrocytes.

The Effect of Short Duration Ultrasound Pulses on the Interaction Between Individual Microbubbles and Fibrin Clots.

In previous work, we examined microscale interactions between microbubbles and fibrin clots under exposure to 1 ms ultrasound pulses. This provided direct evidence that microbubbles were capable of deforming clot boundaries and penetrating into clots, while also affecting fluid uptake and inducing fibrin network damage. Here, we investigate the effect of short duration (15 µs) pulses on microscale bubble-clot interactions as function of bubble diameter (3-9 µm) and pressure. Individual microbubbles (n = 45) were placed at the clot boundary with optical tweezers and exposed to 1 MHz ultrasound. High-speed (10 kfps) imaging and 2-photon microscopy were performed during and after exposure, respectively. While broadly similar phenomena were observed as in the 1 ms pulse case (i.e., bubble penetration, network damage and fluid uptake), substantial quantitative differences were present. The pressure threshold for bubble penetration was increased from 0.39 MPa to 0.6 MPa, and those bubbles that did enter clots had reduced penetration depths and were associated with less fibrin network damage and nanobead uptake. This appeared to be due in large part to increased bubble shrinkage relative to the 1 ms pulse case. Stroboscopic imaging was performed on a subset of bubbles (n = 11) and indicated that complex bubble oscillations can occur during this process.

In situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging.

Converting nanoparticles or monomeric compounds into larger supramolecular structures by endogenous or external stimuli is increasingly popular because these materials are useful for imaging and treating diseases. However, conversion of microstructures to nanostructures is less common. Here, we show the conversion of microbubbles to nanoparticles using low-frequency ultrasound. The microbubble consists of a bacteriochlorophyll-lipid shell around a perfluoropropane gas. The encapsulated gas provides ultrasound imaging contrast and the porphyrins in the shell confer photoacoustic and fluorescent properties. On exposure to ultrasound, the microbubbles burst and form smaller nanoparticles that possess the same optical properties as the original microbubble. We show that this conversion is possible in tumour-bearing mice and could be validated using photoacoustic imaging. With this conversion, our microbubble can potentially be used to bypass the enhanced permeability and retention effect when delivering drugs to tumours.

The influence of compliant boundary proximity on the fundamental and subharmonic emissions from individual microbubbles.

The proximity of a solid-liquid boundary has been theoretically predicted to affect nonlinear microbubble emissions, but to date there has been no experimental validation of this effect. In this study, individual microbubbles (n = 15) were insonicated at f = 11 MHz as a function of offset distance from a compliant (agarose) planar boundary by employing an optical trapping apparatus. It was found that fundamental scattering increases while subharmonic scattering decreases as the microbubble approaches the boundary. Although a microbubble-boundary model can predict the qualitative trends observed for a subset of encapsulation properties, further modeling efforts are required to completely model compliant boundary-microbubble interactions.

Interactions between individual ultrasound-stimulated microbubbles and fibrin clots.

The use of ultrasound-stimulated microbubbles (USMBs) to promote thrombolysis is well established, but there remains considerable uncertainty about the mechanisms of this process. Here we examine the microscale interactions between individual USMBs and fibrin clots as a function of bubble size, exposure conditions and clot type. Microbubbles (n = 185) were placed adjacent to clot boundaries ("coarse" or "fine") using optical tweezers and exposed to 1-MHz ultrasound as a function of pressure (0.1-0.39 MPa). High-speed (10 kfps) imaging was employed, and clots were subsequently assessed with 2-photon microscopy. For fine clots, 46% of bubbles "embedded" within 10 µm of the clot boundary at pressures of 0.1 and 0.2 MPa, whereas at 0.39 MPa, 53% of bubbles penetrated and transited into the clots with an incidence inversely related to their diameter. A substantial fraction of penetrating bubbles induced fibrin network damage and promoted the uptake of nanobeads. In coarse clots, penetration occurred more readily and at lower pressures than in fine clots. The results therefore provide direct evidence of therapeutically relevant effects of USMBs and indicate their dependence on size, exposure conditions and clot properties.

Scaling of the viscoelastic shell properties of phospholipid encapsulated microbubbles with ultrasound frequency.

Phospholipid encapsulated microbubbles are widely employed as clinical diagnostic ultrasound contrast agents in the 1-5 MHz range, and are increasingly employed at higher ultrasound transmit frequencies. The stiffness and viscosity of the encapsulating "shells" have been shown to play a central role in determining both the linear and nonlinear response of microbubbles to ultrasound. At lower frequencies, recent studies have suggested that shell properties can be frequency dependent. At present, there is only limited knowledge of how the viscoelastic properties of phospholipid shells scale at higher frequencies. In this study, four batches of in-house phospholipid encapsulated microbubbles were fabricated with decreasing volume-weighted mean diameters of 3.20, 2.07, 1.82 and 1.61 µm. Attenuation experiments were conducted in order to assess the frequency-dependent response of each batch, resulting in resonant peaks in response at 4.2, 8.9, 12.6 and 19.5 MHz, respectively. With knowledge of the size measurements, the attenuation spectra were then fitted with a standard linearized bubble model in order to estimate the microbubble shell stiffness Sp and shell viscosity Sf, resulting in a slight increase in Sp (1.53-1.76 N/m) and a substantial decrease in Sf (0.29×10(-6)-0.08×10(-6) kg/s) with increasing frequency. These results performed on a single phospholipid agent show that frequency dependent shell properties persist at high frequencies (up to 19.5MHz).

The effect of boundary proximity on the response of individual ultrasound contrast agent microbubbles.

The effect of boundary proximity on ultrasound contrast agent microbubble emissions can play an important role in the context of both targeted microbubble imaging and contrast imaging of microvascular perfusion. In this study, individual microbubbles (n = 104) were insonicated as a function of distance from either a polystyrene membrane (Opticell(TM)) or a compliant agarose boundary up to offset distances of 1000 µm by use of an optical trapping setup. An 'acoustic spectroscopy' approach was employed, which entailed transmitting a sequence of tone bursts with centre frequencies ranging from 4 to 13.5 MHz to determine the frequency and amplitude of maximum radial response (fMR and AMR, respectively). For the Opticell(TM) case, microbubble response exhibited a distinctly oscillatory pattern with increasing offset distance, with an average maximal change in peak frequency and scattered pressure amplitude of 29.6% and 73.2%, respectively, as compared to their values adjacent to the boundary. For the agarose case, microbubbles exhibited an increase in fMR and a decrease in AMR with respect to their values in free space. Simulations indicate the oscillatory dependence on Opticell(TM) distance stems from wavelength-dependent interference phenomena. A recent analytical bubble-boundary model is in broad agreement with the relative AMR changes due to the more compliant agarose layer, however underestimates the change in relative fMR at the boundary.

Methylene blue microbubbles as a model dual-modality contrast agent for ultrasound and activatable photoacoustic imaging.

Ultrasound and photoacoustic imaging are highly complementary modalities since both use ultrasonic detection for operation. Increasingly, photoacoustic and ultrasound have been integrated in terms of hardware instrumentation. To generate a broadly accessible dual-modality contrast agent, we generated microbubbles (a standard ultrasound contrast agent) in a solution of methylene blue (a standard photoacoustic dye). This MB2 solution was formed effectively and was optimized as a dual-modality contrast solution. As microbubble concentration increased (with methylene blue concentration constant), photoacoustic signal was attenuated in the MB2 solution. When methylene blue concentration increased (with microbubble concentration held constant), no ultrasonic interference was observed. Using an MB2 solution that strongly attenuated all photoacoustic signal, high powered ultrasound could be used to burst the microbubbles and dramatically enhance photoacoustic contrast (>800-fold increase), providing a new method for spatiotemporal control of photoacoustic signal generation.

Real-time guidance of thermal and ultrashort pulsed laser ablation in hard tissue using inline coherent imaging.

BACKGROUND AND OBJECTIVE: During tissue ablation, laser light can be delivered with high precision in the transverse dimensions but final incision depth can be difficult to control. We monitor incision depth as it progresses, providing feedback to ensure that material removal occurs within a localized target volume, reducing the possibility of undesirable damage to tissues below the incision. MATERIALS AND METHODS: Ex vivo cortical and cancellous bone was ablated using pulsed lasers with center wavelengths of 1,064 and 1,070 nm, while being imaged in real-time using inline coherent imaging (ICI) at rates of up to 300 kHz and axial resolution of ∼6 µm. With real-time feedback, laser exposure was terminated before perforating into natural inclusions of the cancellous bone and verified by brightfield microscopy of the crater cross-sections accessed via side-polishing. RESULTS: ICI provides direct information about incision penetration even in the presence of intense backscatter from the pulsed laser and plasma emissions. In this study, ICI is able to anticipate structures 176 ± 8 µm below the ablation front with signal intensity 9 ± 2 dB above the noise floor. As a result, the operator is able to terminate exposure of the laser sparing a 50 µm thick layer of bone between the bottom of the incision to a natural inclusion in the cancellous bone. Versatility of the ICI system was demonstrated over a wide range of light-tissue interactions from thermal regime to direct solid-plasma transition. CONCLUSIONS: ICI can be used as non-contact real-time feedback to monitor the depth of an incision created by laser ablation, especially in heterogeneous tissue where ablation rate is less predictable. Furthermore, ICI can image below the ablation front making it possible to stop laser exposure to limit unintentional damage to subsurface structures such as blood vessels or nervous tissue.

In situ 24 kHz coherent imaging of morphology change in laser percussion drilling.

We observe sample morphology changes in real time (24 kHz) during and between percussion drilling pulses by integrating a low-coherence microscope into a laser micromachining platform. Nonuniform cut speed and sidewall evolution in stainless steel are observed to strongly depend on assist gas. Interpulse morphology relaxation such as hole refill is directly imaged, showing dramatic differences in the material removal process dependent on pulse duration/peak power (micros/0.1 kW, ps/20 MW) and material (steel, lead zirconate titanate PZT). Blind hole depth precision is improved by over 1 order of magnitude using in situ feedback from the imaging system.