Monitoring of blood coagulation with non-contact drop oscillation rheometry.

BACKGROUND: Thromboelastography is widely used as a tool to assess the coagulation status of critical-care patients. It allows observation of changes in the material properties of whole blood brought about by clot formation and clot lysis. However, contact activation of the coagulation cascade at surfaces of thromboelastographic systems leads to inherent variability and unreliability in predicting bleeding or thrombosis risks, while also requiring large sample volumes. OBJECTIVES: To develop a non-contact drop oscillation rheometry (DOR) method to measure the viscoelastic properties of blood clots and to compare the results with current laboratory standard measurements. METHODS: Drops of human blood and plasma (5-10 µL) were acoustically levitated. Acoustic field modulation induced drop shape oscillations, and the viscoelastic properties of the sample were calculated by measuring the resonance frequency and damping ratio. RESULTS: DOR showed sensitivity to coagulation parameters. An increase in platelet count resulted in an increase in the maximum clot stiffness. An increase in the calcium ion level enhanced the coagulation rate prior to saturation. An increase in hematocrit resulted in a higher rate of clot formation and increased clot stiffness. Comparison of the results with those obtained with thromboelastography showed that coagulation started sooner with DOR, but with a lower rate and lower maximum stiffness. CONCLUSIONS: DOR can be used as a monitoring tool to assess blood coagulation status. The advantages of small sample size, the lack of contact and small strain (linear viscoelasticity) makes this technique unique for real-time monitoring of blood coagulation.

Fibrin-Targeted Polymerized Shell Microbubbles as Potential Theranostic Agents for Surgical Adhesions.

The development of new therapies for surgical adhesions has proven to be difficult as there is no consistently effective way to assess treatment efficacy in clinical trials without performing a second surgery, which can result in additional adhesions. We have developed lipid microbubble formulations that use a short peptide sequence, CREKA, to target fibrin, the molecule that forms nascent adhesions. These targeted polymerized shell microbubbles (PSMs) are designed to allow ultrasound imaging of early adhesions for diagnostic purposes and for evaluating the success of potential treatments in clinical trials while acting as a possible treatment. In this study, we show that CREKA-targeted microbubbles preferentially bind fibrin over fibrinogen and are stable for long periods of time (∼48 h), that these bound microbubbles can be visualized by ultrasound, and that neither these lipid-based bubbles nor their diagnostic-ultrasound-induced vibrations damage mesothelial cells in vitro. Moreover, these bubbles show the potential to identify adhesionlike fibrin formations and may hold promise in blocking or breaking up fibrin formations in vivo.

Shape oscillations of single blood drops: applications to human blood and sickle cell disease.

Sickle cell disease (SCD) is an inherited blood disorder associated with severe anemia, vessel occlusion, poor oxygen transport and organ failure. The presence of stiff and often sickle-shaped red blood cells is the hallmark of SCD and is believed to contribute to impaired blood rheology and organ damage. Most existing measurement techniques of blood and red blood cell physical properties require sample contact and/or large sample volume, which is problematic for pediatric patients. Acoustic levitation allows rheological measurements in a single drop of blood, simultaneously eliminating the need for both contact containment and manipulation of samples. The technique shows that the shape oscillation of blood drops is able to assess blood viscosity in normal and SCD blood and demonstrates an abnormally increased viscosity in SCD when compared with normal controls. Furthermore, the technique is sensitive enough to detect viscosity changes induced by hydroxyurea treatment, and their dependence on the total fetal hemoglobin content of the sample. Thus this technique may hold promise as a monitoring tool for assessing changes in blood rheology in sickle cell and other hematological diseases.

Small volume method for drug release screening using ultrasonic agitation.

Drug release testing plays a major role along all parts of the dosage form development and manufacturing process. However, official methods to perform this type of testing are often resource intensive and require highly specialized facilities. Affordable and accessible methods for studying drug release behavior are currently lacking. This work presents a small volume approach to solid dissolution and drug release testing of solid dosage forms using ultrasonic agitation. Cavitation and acoustic streaming were generated by a microprobe horn delivering a 40 kHz acoustic signal into a 50 mL test vessel. These two phenomena resulted in breakdown of and release of drug from tablet samples. Prednisone Performance Verification Tablets were used as model tablets to study the effect of system parameters on the drug release process. The effects of these parameters on the acousto-hydrodynamic environment were studied using streak photography and hydrophone measurements. Drug release behavior showed a slow/fast threshold transition separated by a highly variable regime as a function of the system parameters. Observations from drug release experiments and results from acoust-hydrodynamic characterization experiments suggested that this transition is dominated by acoustic streaming. This method represents a screening method to probe relative differences in dosage form composition and acts as a complimentary approach to official testing methods. The small volume format of this test has potential applications in the study of drug release properties from low-dose and novel solid dosage forms as well as reduced cost and increased accessibility of release testing for post-manufacturing tablet quality screening, a current need in low- and middle-income countries.

Enhanced tagging of light utilizing acoustic radiation force with speckle pattern analysis.

In optical imaging, the depth and resolution are limited due to scattering. Unlike light, scattering of ultrasound (US) waves in tissue is negligible. Hybrid imaging methods such as US-modulated optical tomography (UOT) use the advantages of both modalities. UOT tags light by inducing phase change caused by modulating the local index of refraction of the medium. The challenge in UOT is detecting the small signal. The displacement induced by the acoustic radiation force (ARF) is another US effect that can be utilized to tag the light. It induces greater phase change, resulting in a stronger signal. Moreover, the absorbed acoustic energy generates heat, resulting in change in the index of refraction and a strong phase change. The speckle pattern is governed by the phase of the interfering scattered waves; hence, speckle pattern analysis can obtain information about displacement and temperature changes. We have presented a model to simulate the insonation processes. Simulation results based on fixed-particle Monte Carlo and experimental results show that the signal acquired by utilizing ARF is stronger compared to UOT. The introduced mean irradiance change (MIC) signal reveals both thermal and mechanical effects of the focused US beam in different timescales. Simulation results suggest that variation in the MIC signal can be used to generate a displacement image of the medium.

Outcomes of the collapse of a large bubble in water at high ambient pressures.

Presented here are observations of the outcomes of the collapses of large single bubbles in H_{2}O and D_{2}O at high ambient pressures. Experiments were carried out in a high-pressure spherical resonator at ambient pressures of up to 30 MPa and acoustic pressures up to 35 MPa. Monitoring of the collapse events and their outcomes was accomplished using multiframe high-speed photography. Among the observations to be presented are the temporal and spatial evolution of light emissions produced by the collapse events, which were observed to last on the order of 30 ns and have time independent radii on the order of 30µm; the production of Rayleigh-Taylor jets which were observed to travel distances of up to 70µm at speeds in excess of 4500 m/s; the entrainment of the light emitting regions in the jets' remnants; the production of spheroidal objects around the collapse points of the bubbles, far from any surface of the resonator; and the traversal and emergence of the Rayleigh-Taylor jets through the spherical objects. These spheroidal objects appear to behave as amorphous solids and form at locations where hydrodynamics predicts pressures in excess of the known transition pressures of water into the high-pressure crystalline ices, Ice-VI and Ice-VII.

Microvessels-on-a-Chip to Assess Targeted Ultrasound-Assisted Drug Delivery.

Microbubbles have been used in ultrasound-assisted drug delivery to help target solid tumors via blood vessels in vivo; however, studies to understand the phenomena at the cellular level and to optimize parameters for ultrasound or microbubbles in vivo are challenging and expensive to perform. Here, we utilize microfluidic microvessels-on-a-chip that enable visualization of microbubble/ultrasound-dependent drug delivery to microvasculature. When exposed to pulsed ultrasound, microbubbles perfused through microvessels-on-a-chip were observed to stably oscillate. Minimal cellular damage was observed for both microbubbles and untargeted doxorubicin-encapsulating liposomes (DOX-liposomes) perfused through chip microvessels. In contrast, passive and ultrasound-assisted perfusion of integrin-targeted DOX-liposomes induced cytotoxicity, which was only significantly enhanced for ultrasound-assisted perfusion when microbubbles were coperfused. These results suggest that stably oscillating microbubbles enhance targeted DOX-liposome internalization/cytotoxicity largely by stimulating integrin receptor endocytosis. Furthermore, our study demonstrates the utility of our microvessels-on-a-chip as a screening platform for optimizing drug dosage, targeting ligands and drugs.

Temporally and spatially resolved imaging of laser-nucleated bubble cloud sonoluminescence.

Imaging techniques have been used to capture the temporal and spatial evolution of light emissions from collapsing bubble clouds at high static pressures. Emission events lasting up to 70 ns with peak diameters nearing 1 mm have been observed. Observations of the cloud evolution before and after emission events have been made. Photomultiplier tube monitoring has been employed in conjunction with imaging to study the temporal characteristics of light emission.

Ambient acceleration dependence of single-bubble sonoluminescence.

Much of the research performed to study SBSL deals with the influence of external parameters (e.g., the host water temperature, the ambient pressure, the type and amount of dissolved gas in the liquid, to name a few) on the bubble dynamics and light emission. In the current paper, work carried out to study the influence of another external parameter-ambient acceleration-is described. The experiments described here were performed on the NASA KC-135 which provided both periods of reduced gravity (10(-3) g) and increased gravity (1.8 g) by flying repeated parabolic maneuvers. The resulting measurements are compared with the predictions of a numerical model and can be understood in the context of the changing hydrostatic head pressure and buoyant force acting on the bubble.

Suppressing shape instabilities to discover the Bjerknes force instability (L).

For sufficiently strong acoustic forcing in a standing wave field, subresonant size bubbles are predicted to be repelled from the pressure antinode. Single bubble sonoluminescence (SBSL) conditions in water do not allow the observation of this instability. This study investigates the possibility that increasing the viscosity of the host liquid can preferentially suppress shape instabilities of a bubble and allow SBSL experiments to be limited by the Bjerknes force instability.

Transient cavitation in high-quality-factor resonators at high static pressures.

It is well known that cavitation collapse can generate intense concentrations of mechanical energy, sufficient to erode even the hardest metals and to generate light emissions visible to the naked eye [sonoluminescence (SL)]. Considerable attention has been devoted to the phenomenon of "single bubble sonoluminescence" (SBSL) in which a single stable cavitation bubble radiates light flashes each and every acoustic cycle. Most of these studies involve acoustic resonators in which the ambient pressure is near 0.1 MPa (1 bar), and with acoustic driving pressures on the order of 0.1 MPa. This study describes a high-quality factor, spherical resonator capable of achieving acoustic cavitation at ambient pressures in excess of 30 MPa (300 bars). This system generates bursts of violent inertial cavitation events lasting only a few milliseconds (hundreds of acoustic cycles), in contrast with the repetitive cavitation events (lasting several minutes) observed in SBSL; accordingly, these events are described as "inertial transient cavitation." Cavitation observed in this high pressure resonator is characterized by flashes of light with intensities up to 1000 times brighter than SBSL flashes, as well as spherical shock waves with amplitudes exceeding 30 MPa at the resonator wall. Both SL and shock amplitudes increase with static pressure.

Detecting cavitation in mercury exposed to a high-energy pulsed proton beam.

The Oak Ridge National Laboratory Spallation Neutron Source employs a high-energy pulsed proton beam incident on a mercury target to generate short bursts of neutrons. Absorption of the proton beam produces rapid heating of the mercury, resulting in the formation of acoustic shock waves and the nucleation of cavitation bubbles. The subsequent collapse of these cavitation bubbles promote erosion of the steel target walls. Preliminary measurements using two passive cavitation detectors (megahertz-frequency focused and unfocused piezoelectric transducers) installed in a mercury test target to monitor cavitation generated by proton beams with charges ranging from 0.041 to 4.1 muC will be reported on. Cavitation was initially detected for a beam charge of 0.082 muC by the presence of an acoustic emission approximately 250 mus after arrival of the incident proton beam. This emission was consistent with an inertial cavitation collapse of a bubble with an estimated maximum bubble radius of 0.19 mm, based on collapse time. The peak pressure in the mercury for the initiation of cavitation was predicted to be 0.6 MPa. For a beam charge of 0.41 muC and higher, the lifetimes of the bubbles exceeded the reverberation time of the chamber ( approximately 300 mus), and distinct windows of cavitation activity were detected, a phenomenon that likely resulted from the interaction of the reverberation in the chamber and the cavitation bubbles.

Sensing a buried resonant object by single-channel time reversal.

Scaled laboratory experiments are conducted to assess the efficacy of iterative, single-channel time reversal for enhancement of monostatic returns from resonant spheres in the free field and buried in a sediment phantom. Experiments are performed in a water tank using a broad-band piston transducer operating between 0.4 and 1.5 MHz and calibrated using free surface reflections. Solid and hollow metallic spheres, 6.35 mm in diameter, are buried in a consolidation of 128-microm-mean- diameter spherical glass beads. The procedure consists of exciting the target object with a broadband pulse, sampling the return using a finite time window, reversing the signal in time, and using this reversed signal as the source waveform for the next interrogation. Results indicate that the spectrum of the returns rapidly converges to the dominant mode in the backscattering response of the target. Signal-to-noise enhancement of the target echo is demonstrated for a target at several burial depths. Images generated by scanning the transducer over the location of multiple buried targets demonstrate the ability of the technique to distinguish between targets of differing type and to yield an enhancement of different modes within the response of a single target as a function of transducer position and processing bandwidth.

Temporal and spatial detection of HIFU-induced inertial and hot-vapor cavitation with a diagnostic ultrasound system.

The onset and presence of inertial cavitation and near-boiling temperatures in high-intensity focused ultrasound (HIFU) therapy have been identified as important indicators of energy deposition for therapy guidance. Passive cavitation detection is commonly used to detect bubble emissions, where a fixed-focus single-element acoustic transducer is typically used as a passive cavitation detector (PCD). This technique is suboptimal for clinical applications, because most PCD transducers are tightly focused and afford limited spatial coverage of the HIFU focal region. A Terason 2000 Ultrasound System was used as a PCD array to expand the spatial detection region for cavitation by operating in passive mode, obtaining the radiofrequency signals corresponding to each scan line and filtering the contribution from scattering of the HIFU signal harmonics. This approach allows for spatially resolved detection of both inertial and stable cavitation throughout the focal region. Measurements with the PCD array during sonication with a 1.1-MHz HIFU source in tissue phantoms were compared with single-element PCD and thermocouple sensing. Stable cavitation signals at the harmonics and superharmonics increased in a threshold fashion for temperatures >90 degrees C, an effect attributed to high vapor pressure in the cavities. Incorporation of these detection techniques in a diagnostic ultrasound platform could result in a powerful tool for improving HIFU guidance and treatment.

Design of a system to measure light scattering from individual cells excited by an acoustic wave.

A system to measure light scattering from individual cells excited by an acoustic wave was designed, and tests were performed on live Jurkat cells. Cells passing in a laminar stream within a water bath were excited by a focused ultrasound pulse, while the scattered light from a laser beam was monitored at various scattering angles. The cells were modeled as viscoelastic liquid drops, which return to equilibrium via shape oscillations after an acoustically-induced deformation. The Fast Fourier Transform of the scattered light signal was used to extract information about the highly-damped resonant frequencies of the cells, and the detected frequencies are consistent with theoretical predictions.

Experimental validation of a tractable numerical model for focused ultrasound heating in flow-through tissue phantoms.

Heating from high intensity focused ultrasound (HIFU) can be used to control bleeding, both from individual blood vessels as well as from gross damage to the capillary bed. The presence of vascularity can limit one's ability to elevate the temperature owing to convective heat transport. In an effort to better understand the heating process in tissues with vascular structure we have developed a numerical simulation that couples models for ultrasound propagation, acoustic streaming, ultrasound heating and blood cooling in a Newtonian viscous medium. The 3-D simulation allows for the study of complicated biological structures and insonation geometries. We have also undertaken a series of in vitro experiments employing non-uniform flow-through tissue phantoms and designed to provide verification of the model predictions. We show that blood flow of 2 cm/s (6.4 ml/min through a 2.6 mm 'vessel') can reduce peak temperature in a vessel wall by 25%. We also show that HIFU intensities of 6.5 x 10(5) W/m2 can induce acoustic streaming with peak velocities up to 5 cm/s and this can reduce heating near a vessel wall by more than 10%. These results demonstrate that convective cooling is important in HIFU and can be accounted for within simulation models.

Bubble dynamics near the onset of single-bubble sonoluminescence.

A number of groups have reported measurements of the location in the parameter space of bubble size versus acoustic pressure amplitude of shape- and size-stable bubbles. For air/water systems, a general trend emerges: stable bubbles are found on one of two line paths in this space defined by their range of acoustic pressure. Bubbles on the higher-pressure path emit light. There have been few studies of the transition between these two paths. In this work we describe our observations of this transition regime. In this regime, a slow time scale oscillation (period 2-7 s) in the bubble size, position, and phase of flash timing can be observed. At lower dissolved gas concentrations, a hysteresis in the bubble size as a function of acoustic pressure is observed, complementing previous light intensity measurements reported in the literature.

Bubble dynamics and size distributions during focused ultrasound insonation.

The deposition of ultrasonic energy in tissue can cause tissue damage due to local heating. For pressures above a critical threshold, cavitation will occur, inducing a much larger thermal energy deposition in a local region. The present work develops a nonlinear bubble dynamics model to numerically investigate bubble oscillations and bubble-enhanced heating during focused ultrasound (HIFU) insonation. The model is applied to calculate two threshold-dependent phenomena occurring for nonlinearly oscillating bubbles: Shape instability and growth by rectified diffusion. These instabilities in turn are shown to place physical boundaries on the time-dependent bubble size distribution, and thus the thermal energy deposition.

Acoustical stability of a sonoluminescing bubble.

In the parameter region for sonoluminescence of a single levitated bubble in a water-filled resonator it is observed that the bubble may have an enormous spatial stability leaving it "pinned" in the fluid and allowing it to emit light pulses of picosecond accuracy. We report here observations of a complex harmonic structure in the acoustic field surrounding a sonoluminescing bubble. We show that this complex sound field determines the position of the bubble and may either increase or decrease its spatial stability. The acoustic environment of the bubble is the result of the excitation of high-order normal modes of the resonator by the outgoing shock wave generated by the bubble collapse.