Estimation of fat content in soft tissues using dual frequency ultrasound-A phantom study.

This paper presents an initial investigation into the use of dual frequency pulse-echo ultrasound, second order ultrasound field (SURF) imaging, to measure the fat content of soft tissues. The SURF imaging method was used to measure the non-linear bulk elasticity (NBE) of several fatty phantoms that were created by mixing different mass fractions of soybean oil uniformly into agar phantoms. The median of the measured NBE within the estimation region was found to increase linearly with fat mass fraction (R2 = 0.99), from 1.7 GPa-1 at 9.6% fat to 2.52 GPa-1 at 63.6% fat, thus, showing promise as a sensitive parameter for fat content measurement. Comparisons to mixture laws in earlier literature are made, and the most important error sources that need to be considered for the in vivo applications of the method are discussed.

Enhancing carrier flux for efficient drug delivery in cancer tissues.

Ultrasound focused toward tumors in the presence of circulating microbubbles improves the delivery of drug-loaded nanoparticles and therapeutic outcomes; however, the efficacy varies among the different properties and conditions of the tumors. Therefore, there is a need to optimize the ultrasound parameters and determine the properties of the tumor tissue important for the successful delivery of nanoparticles. Here, we propose a mesoscopic model considering the presence of entropic forces to explain the ultrasound-enhanced transport of nanoparticles across the capillary wall and through the interstitium of tumors. The nanoparticles move through channels of variable shape whose irregularities can be assimilated to barriers of entropic nature that the nanoparticles must overcome to reach their targets. The model assumes that focused ultrasound and circulating microbubbles cause the capillary wall to oscillate, thereby changing the width of transcapillary and interstitial channels. Our analysis provides values for the penetration distances of nanoparticles into the interstitium that are in agreement with experimental results. We found that the penetration increased significantly with increasing acoustic intensity as well as tissue elasticity, which means softer and more deformable tissue (Young modulus lower than 50 kPa), whereas porosity of the tissue and pulse repetition frequency of the ultrasound had less impact on the penetration length. We also considered that nanoparticles can be absorbed into cells and to extracellular matrix constituents, finding that the penetration length is increased when there is a low absorbance coefficient of the nanoparticles compared with their diffusion coefficient (close to 0.2). The model can be used to predict which tumor types, in terms of elasticity, will successfully deliver nanoparticles into the interstitium. It can also be used to predict the penetration distance into the interstitium of nanoparticles with various sizes and the ultrasound intensity needed for the efficient distribution of the nanoparticles.

Effect of Acoustic Radiation Force on the Distribution of Nanoparticles in Solid Tumors.

Acoustic radiation force (ARF) might improve the distribution of nanoparticles (NPs) in tumors. To study this, tumors growing subcutaneously in mice were exposed to focused ultrasound (FUS) either 15 min or 4 h after the injection of NPs, to investigate the effect of ARF on the transport of NPs across the vessel wall and through the extracellular matrix. Quantitative analysis of confocal microscopy images from frozen tumor sections was performed to estimate the displacement of NPs from blood vessels. Using the same experimental exposure parameters, ARF was simulated and compared with the experimental data. Enhanced interstitial transport of NPs in tumor tissues was observed when FUS (10 MHz, acoustic power 234 W/cm2, 3.3% duty cycle) was given either 15 min or 4 h after NP administration. According to acoustic simulations, the FUS generated an ARF per unit volume of 2.0×106 N/m3. The displacement of NPs was larger when FUS was applied 4 h after NP injection compared with after 15 min. This study shows that ARF might contribute to a modest improved distribution of NPs into the tumor interstitium.

Effect of Acoustic Radiation Force on Displacement of Nanoparticles in Collagen Gels.

Penetration of nanoscale therapeutic agents into the extracellular matrix (ECM) of a tumor is a limiting factor for the sufficient delivery of drugs in tumors. Ultrasound (US) in combination with microbubbles causing cavitation is reported to improve delivery of nanoparticles (NPs) and drugs to tumors. Acoustic radiation force (ARF) could also enhance the penetration of NPs in tumor ECM. In this work, a collagen gel was used as a model for tumor ECM to study the effects of ARF on the penetration of NPs as well as the deformation of collagen gels applying different US parameters (varying pressure and duty cycle). The collagen gel was characterized, and the diffusion of water and NPs was measured. The penetration of NPs into the gel was measured by confocal laser scanning microscopy and numerical simulations were performed to determine the ARF and to estimate the penetration distance and extent of deformation. ARF had no effect on the penetration of NPs into the collagen gels for the US parameters and gel used, whereas a substantial deformation was observed. The width of the deformation on the collagen gel surface corresponded to the US beam. Comparing ARF caused by attenuation within the gel and Langevin pressure caused by reflection at the gel-water surface, ARF was the prevalent mechanism for the gel deformation. The experimental and theoretical results were consistent both with respect to the NP penetration and the gel deformation.

Nonlinear bulk elasticity imaging using dual frequency ultrasound.

The nonlinear acoustic bulk properties of tissue, e.g., the coefficient of nonlinearity, ßn, or the nonlinear bulk elasticity, ßp=ßnκ0, have been shown to be promising parameters for tissue characterization due to their sensitivity to tissue structure. Previously developed methods for imaging these parameters using single frequency ultrasound have shown success in a laboratory setting using the transmission mode. In the pulse-echo mode, however, unknown absorption, diffraction, and speckle produce unreliable estimates and instability, causing these methods to have achieved no clinical relevance. In this paper, a pulse-echo method for measurement of the nonlinear bulk elasticity is presented using a dual frequency approach. The method is less sensitive to diffraction and absorption due to a separate low frequency manipulation wave. The technique is tested in both simulations and in vitro in a heterogeneous phantom with two regions of different nonlinear properties. Both in simulations and in vitro, a spatial ßp map is produced where the two regions are clearly distinguished. In addition, the quantitative estimates of ßp obtained are close to the expected values, making the method a promising first step toward in vivo imaging of nonlinear bulk properties.

Exploiting Ballou's rule for better tissue classification.

Ultrasound tissue characterization based on the coefficient of nonlinearity, ßn = 1 + B/2A, has been demonstrated to produce added diagnostic value due to its large variation and sensitivity to tissue structure. However, the parameter has been observed to be significantly correlated to the speed of sound and density. These relationships are analyzed empirically as well as theoretically by developing a pressure-density relation based on a thermodynamic model and the Mie intermolecular potential. The results indicate that for many soft tissues, the coefficient of nonlinearity is largely determined by the isentropic compressibility, κs. Consequently, for tissue characterization, estimating the nonlinear response of the medium, given by ßp = ßnκs, appears to be beneficial due to correlated quantities.

Analysis of acoustic impedance matching in dual-band ultrasound transducers.

Dual-frequency band probes are needed for ultrasound (US) reverberation suppression and are useful for image-guided US therapy. A challenge is to design transducer stacks that achieve high bandwidth and efficiency at both operating frequencies when the frequencies are widely separated with a frequency ratio ∼6:1-20:1. This paper studies the loading and backing conditions of transducers in such stacks. Three stack configurations are presented and analyzed using one-dimensional models. It is shown that a configuration with three layers of material separating the transducers is favorable, as it reduces high frequency ringing by ∼20 dB compared to other designs, and matches the low frequency (LF) transducer to the load at a lower frequency. In some cases, the LF load matching is governed by a simple mass-spring interaction in spite of having a complicated matching structure. The proposed design should yield improved performance of reverberation suppression algorithms. Its suitability for reduction of probe heating, also in single-band probes, should be investigated.

Adaptive reverberation noise delay estimation for reverberation suppression in dual band ultrasound imaging.

The behavior of the propagation delays introduced in dual frequency band ultrasound imaging is discussed. In particular, the delay of reverberation noise components is examined. Using a delay corrected subtraction (DCS) method, it is possible to suppress the reverberation noise if the behavior of the propagation delays is known. Here, a signal adaptive estimation for the reverberation delay is introduced and applied through DCS to suppress reverberation noise in a numerically simulated signal. The reverberation reduction is compared to DCS suppression using a simpler delay estimation and shows that a signal based adaptive estimation yields a improved suppression of reverberation noise. The study indicates that the advantage of the adaptive estimation is highest when the medium has changing nonlinearity with depth.

Ultrasound-enhanced drug delivery in prostate cancer xenografts by nanoparticles stabilizing microbubbles.

The delivery of nanoparticles to solid tumors is often ineffective due to the lack of specificity towards tumor tissue, limited transportation of the nanoparticles across the vascular wall and poor penetration through the extracellular matrix of the tumor. Ultrasound is a promising tool that can potentially improve several of the transportation steps, and the interaction between sound waves and microbubbles generates biological effects that can be beneficial for the successful delivery of nanocarriers and their contents. In this study, a novel platform consisting of nanoparticle-stabilized microbubbles has been investigated for its potential for ultrasound-enhanced delivery to tumor xenografts. Confocal laser scanning microscopy was used to study the supply of nanoparticles from the vasculature and to evaluate the effect of different ultrasound parameters at a microscopic level. The results demonstrated that although the delivery is heterogeneous within tumors, there is a significant improvement in the delivery and the microscopic distribution of both nanoparticles and a released model drug when the nanoparticles are combined with microbubbles and ultrasound. The mechanisms that underlie the improved delivery are discussed.

Methods for reverberation suppression utilizing dual frequency band imaging.

Reverberations impair the contrast resolution of diagnostic ultrasound images. Tissue harmonic imaging is a common method to reduce these artifacts, but does not remove all reverberations. Dual frequency band imaging (DBI), utilizing a low frequency pulse which manipulates propagation of the high frequency imaging pulse, has been proposed earlier for reverberation suppression. This article adds two different methods for reverberation suppression with DBI: the delay corrected subtraction (DCS) and the first order content weighting (FOCW) method. Both methods utilize the propagation delay of the imaging pulse of two transmissions with alternating manipulation pressure to extract information about its depth of first scattering. FOCW further utilizes this information to estimate the content of first order scattering in the received signal. Initial evaluation is presented where both methods are applied to simulated and in vivo data. Both methods yield visual and measurable substantial improvement in image contrast. Comparing DCS with FOCW, DCS produces sharper images and retains more details while FOCW achieves best suppression levels and, thus, highest image contrast. The measured improvement in contrast ranges from 8 to 27 dB for DCS and from 4 dB up to the dynamic range for FOCW.

Ultrasound improves the uptake and distribution of liposomal Doxorubicin in prostate cancer xenografts.

Combining liposomally encapsulated cytotoxic drugs with ultrasound exposure has improved the therapeutic response to cancer in animal models; however, little is known about the underlying mechanisms. This study focused on investigating the effect of ultrasound exposures (1 MHz and 300 kHz) on the delivery and distribution of liposomal doxorubicin in mice with prostate cancer xenografts. The mice were exposed to ultrasound 24 h after liposome administration to study the effect on release of doxorubicin and its penetration through the extracellular matrix. Optical imaging methods were used to examine the effects at both microscopic subcellular and macroscopic tissue levels. Confocal laser scanning microscopy revealed that ultrasound-exposed tumors had increased levels of released doxorubicin compared with unexposed control tumors and that the distribution of liposomes and doxorubicin through the tumor tissue was improved. Whole-animal optical imaging revealed that liposomes were taken up by both abdominal organs and tumors.

Mechanisms of the ultrasound-mediated intracellular delivery of liposomes and dextrans.

The mechanism involved in the ultrasoundenhanced intracellular delivery of fluorescein-isothiocyanate (FITC)-dextran (molecular weight 4 to 2000 kDa) and liposomes containing doxorubicin (Dox) was studied using HeLa cells and an ultrasound transducer at 300 kHz, varying the acoustic power. The cellular uptake and cell viability were measured using flow cytometry and confocal microscopy. The role of endocytosis was investigated by inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis. Microbubbles were found to be required during ultrasound treatment to obtain enhanced cellular uptake. The percentage of cells internalizing Dox and dextran increased with increasing mechanical index. Confocal images and flow cytometric analysis indicated that the liposomes were disrupted extracellularly and that released Dox was taken up by the cells. The percentage of cells internalizing dextran was independent of the molecular weight of dextrans, but the amount of the small 4-kDa dextran molecules internalized per cell was higher than for the other dextrans. The inhibition of endocytosis during ultrasound exposure resulted in a significant decrease in cellular uptake of dextrans. Therefore, the improved uptake of Dox and dextrans may be a result of both sonoporation and endocytosis.

SURF imaging beams in an aberrative medium: Generation and postprocessing enhancement.

This paper presents numerical simulations of dual-frequency second-order ultrasound field (SURF) reverberation suppression transmit-pulse complexes. Such propagation was previously studied in a homogeneous medium. In this work, the propagation path includes a strongly aberrating body wall modeled by a sequence of delay screens. Each of the applied SURF transmit pulse complexes consists of a high-frequency 3.5-MHz imaging pulse combined with a low-frequency 0.5-MHz sound speed manipulation pulse. Furthermore, the feasibility of two signal postprocessing methods are investigated using the aberrated transmit SURF beams. These methods have previously been shown to adjust the depth of maximum SURF reverberation suppression within a homogeneous medium. The need for this study arises because imaging situations in which reverberation suppression is useful are also likely to produce pulse wave front distortion (aberration). Such distortions could potentially produce time delays that cancel the accumulated propagation time delay needed for the SURF reverberation suppression technique. Results show that both the generation of synthetic SURF reverberation suppression imaging transmit beams and the following postprocessing adjustments are attainable even when a body wall introduces time delays which are larger than previously reported delays measured on human body wall specimens.

Effect of ultrasound parameters on the release of liposomal calcein.

The ultrasound exposure parameters that maximize drug release from dierucoyl-phosphatidylcholine (DEPC)-based liposomes were studied using two transducers operating at 300 kHz and 1 MHz. Fluorescent calcein was used as a model drug, and the release from liposomes in solution was measured using a spectrophotometer. The release of calcein was more efficient at 300 kHz than at 1 MHz, with thresholds of peak negative pressures of 0.9 MPa and 1.9 MPa, respectively. Above this threshold, the release increased with increasing peak negative pressure, mechanical index (MI), and duty cycle. The amount of drug released followed first-order kinetics and increased with exposure time to a maximal release. To increase the release further, the MI had to be increased. The results demonstrate that the MI and the overall exposure time are the major parameters that determine the drug's release. The drug's release is probably due to mechanical (cavitation) rather than thermal effects, and that was also confirmed by the detection of hydroxide radicals.

Nonlinear propagation delay and pulse distortion resulting from dual frequency band transmit pulse complexes.

A method of acoustic imaging is discussed that potentially can improve the diagnostic capabilities of medical ultrasound. The method, given the name second order ultrasound field imaging, is achieved by the processing of the received signals from transmitted dual frequency band pulse complexes with at least partly overlapping high frequency (HF) and low frequency (LF) pulses. The transmitted HF pulses are used for image reconstruction whereas the transmitted LF pulses are used to manipulate the elastic properties of the medium observed by the HF imaging pulses. In the present paper, nonlinear propagation effects observed by a HF imaging pulse due to the presence of a LF manipulation pulse is discussed. When using dual frequency band transmit pulse complexes with a large separation in center frequency (e.g., 1:10), these nonlinear propagation effects are manifested as a nonlinear HF propagation delay and a HF pulse distortion different from conventional harmonic distortion. In addition, with different transmit foci for the HF and LF pulses, nonlinear aberration will occur.

Contrast imaging by non-overlapping dual frequency band transmit pulse complexes.

SURF contrast imaging, as described previously in the literature, is a contrast agent detection technique achieved by processing of the received signals from transmitted dual frequency band pulse complexes with overlapping high-frequency (HF) and low-frequency (LF) pulses. The transmitted HF pulses are used for image reconstruction, whereas the transmitted LF pulses are used to manipulate the scattering properties of the contrast agent. As with harmonic contrast agent detection techniques, nonlinear wave propagation will, in most situations, also limit the specificity with the SURF contrast technique when transmitting overlapping HF and LF pulses. The present paper proposes an alternative SURF contrast imaging technique using transmit dual frequency band pulse complexes with non-overlapping HF and LF pulses. If the frequency of the LF manipulation pulse is close to the bubble resonance frequency, numerical simulations indicate a significant ring-down effect of the LF bubble radius response. Utilizing this bubble ring-down effect and transmitting the HF pulse just after the LF pulse, a contrast agent specificity approaching infinity accompanied by a contrast agent sensitivity only for contrast bubbles having resonance frequencies within a narrow frequency range may be obtained.

Post-processing enhancement of reverberation-noise suppression in dual-frequency SURF imaging.

A post-processing adjustment technique to enhance dual-frequency second-order ultrasound field (SURF) reverberation-noise suppression imaging in medical ultrasound is analyzed. Two variant methods are investigated through numerical simulations. They both solely involve post-processing of the propagated high-frequency (HF) imaging wave fields, which in real-time imaging corresponds to post-processing of the beamformed receive radio-frequency signals. Hence, the transmit pulse complexes are the same as for the previously published SURF reverberation-suppression imaging method. The adjustment technique is tested on simulated data from propagation of SURF pulse complexes consisting of a 3.5-MHz HF imaging pulse added to a 0.5-MHz low-frequency soundspeed manipulation pulse. Imaging transmit beams are constructed with and without adjustment. The post-processing involves filtering, e.g., by a time-shift, to equalize the two SURF HF pulses at a chosen depth. This depth is typically chosen to coincide with the depth where the first scattering or reflection occurs for the reverberation noise one intends to suppress. The beams realized with post-processing show energy decrease at the chosen depth, especially for shallow depths where, in a medical imaging situation, a body-wall is often located. This indicates that the post-processing may further enhance the reverberation- suppression abilities of SURF imaging. Moreover, it is shown that the methods might be utilized to reduce the accumulated near-field energy of the SURF transmit-beam relative to its imaging region energy. The adjustments presented may therefore potentially be utilized to attain a slightly better general suppression of multiple scattering and multiple reflection noise compared with non-adjusted SURF reverberation-suppression imaging.

Nonlinear propagation acoustics of dual-frequency wide-band excitation pulses in a focused ultrasound system.

In this article, acoustic propagation effects of dual-frequency wide-band excitation pulses in a focused ultrasound system are demonstrated in vitro. A designed and manufactured dual-frequency band annular array capable of transmitting 0.9/7.5 MHz center frequency wide-band pulses was used for this purpose. The dual-frequency band annular array, has been designed using a bi-layer piezo-electric stack. Water tank measurements demonstrate the function of the array by activating the low- and high-frequency layers individually and simultaneously. The results show that the array works as intended. Activating the low- and high-frequency layers individually, results in less than -50 dB signal level from the high- and low-frequency layers respectively. Activating both layers simultaneously, produce a well defined dual-frequency pulse. The presence of the low-frequency pulse leads to compression, expansion, and a time delay of the high-frequency pulse. There is a phase shift between the low- and high-frequency pulse as it propagates from the array to the focus. This makes the latter described effects also dependent on the array configuration. By varying the low-frequency pressure, a shift of up to 0.5 MHz in center frequency of a 8.0 MHz transmitted high-frequency pulse is observed at the array focus. The results demonstrate the high propagation complexity of dual-frequency pulses.

Utilizing dual frequency band transmit pulse complexes in medical ultrasound imaging.

A method of acoustic imaging that potentially can improve the diagnostic capabilities of medical ultrasound is presented. The method, given the name SURF (Second order UltRasound Field) imaging, is achieved by processing the received signals from transmitted dual frequency band pulse complexes with at least partly overlapping high frequency (HF) and low frequency (LF) pulses. The transmitted HF pulses are used for image reconstruction, whereas the transmitted LF pulses are used to manipulate the elastic properties of the medium observed by the HF imaging pulses. The present paper discusses fundamental concepts in relation to the use of dual frequency band pulse complexes for medical ultrasound imaging.

Transmit beams adapted to reverberation noise suppression using dual-frequency SURF imaging.

A method that uses dual-frequency pulse complexes of widely separated frequency bands to suppress noise caused by multiple scattering or multiple reflections in medical ultrasound imaging is presented. The excitation pulse complexes are transmitted to generate a second order ultrasound field (SURF) imaging synthetic transmit beam. This beam has reduced amplitude near the transducer, which illustrates the multiple scattering suppression ability of the imaging method. Field simulations solving a nonlinear wave equation are used to calculate SURF imaging beams, which are compared with beams for pulse inversion (PI) and fundamental imaging. In addition, a combined SURF and PI beam generation is described and compared with the beams mentioned above. A quality ratio, relating the energy within the near-field to that within the imaging region, is defined and used to score the multiple scattering and multiple reflection suppression abilities when imaging with the different beams. The realized combined SURF-PI beam scores highest, followed by SURF, PI (that score equally well), and the fundamental. The amplitude in the imaging region and therefore also the SNR is highest for the fundamental followed by SURF, PI, and SURF-PI. The work hence indicates that when substituting PI for SURF, one may trade increased SNR into use of increased imaging frequencies without loss of multiple scattering and multiple reflection noise suppression.