Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos.

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

Nonlinear contrast imaging with an array-based micro-ultrasound system.

The main goal of this study was to determine the optimal strategy for a real-time nonlinear contrast mode for small-animal imaging at high frequencies, on a new array-based micro-ultrasound system. Previously reported contrast imaging at frequencies above 15 MHz has primarily relied on subtraction schemes involving B-mode image data. These approaches provide insufficient contrast to tissue ratios under many imaging conditions. In this work, pulse inversion, amplitude modulation and combinations of these were systematically investigated for the detection of nonlinear fundamental and subharmonic signal components to maximize contrast-to-tissue ratio (CTR) in the 18-24 MHz range. From in vitro and in vivo measurements, nonlinear fundamental detection with amplitude modulation provided optimal results, allowing an improvement in CTR of 13 dB compared with fundamental imaging. Based on this detection scheme, in vivo parametric images of murine kidneys were generated using sequences of nonlinear contrast images after intravenous bolus injections of microbubble suspensions. Initial parametric images of peak enhancement (PE), wash-in rate (WiR) and rise time (RT) are presented. The parametric images are indicative of blood perfusion kinetics, which, in the context of preclinical imaging with small animals, are anticipated to provide valuable insights into the progression of human disease models, where blood perfusion plays a critical role in either the diagnosis or treatment of the disease.

A method for differentiating targeted microbubbles in real time using subharmonic micro-ultrasound and interframe filtering.

This study introduces a new method for differentiating targeted microbubbles in the presence of flowing microbubbles and tissue using micro-ultrasound. The method relies on subharmonic (SH) imaging for segmenting microbubble signals from tissue signals, and low-pass interframe filtering for segmenting bound targeted microbubbles from flowing microbubbles. The method is evaluated with 30 frames per second SH B-mode imaging in vitro, using a wall-less vessel flow phantom. The SH B-mode cineloops were postprocessed using an interframe moving average filter to segment the regions of bound microbubbles on the inner surface of the vessel phantom. The bound bubbles were then disrupted with sufficiently high ultrasound pressures, so that the dynamic process of targeted microbubble binding under flowing conditions could be observed. These preliminary results show that the proposed method is a feasible solution to the challenge of differentiating targeted microbubbles in the presence of tissue and freely flowing microbubbles at high frequencies, which in turn should improve the specificity of targeted microbubble detection.

High-frequency subharmonic pulsed-wave Doppler and color flow imaging of microbubble contrast agents.

A recent study has shown the feasibility of subharmonic (SH) flow imaging at a transmit frequency of 20 MHz. This paper builds on these results by examining the performance of SH flow imaging as a function of transmit pressure. Further, we also investigate the feasibility of SH pulsed-wave Doppler (PWD) imaging. In vitro flow experiments were performed with a 1-mm-diameter wall-less vessel cryogel phantom using the ultrasound contrast agent Definity and an imaging frequency of 20 MHz. The phantom results show that there is an identifiable pressure range where accurate flow velocity and power estimates can be made with SH imaging at 10 MHz (SH10), above which velocity estimates are biased by radiation force effects and unstable bubble behavior, and below which velocity and power estimates are degraded by poor SNR. In vivo validation of SH PWD was performed in an arteriole of a rabbit ear, and blood velocity estimates compared well with fundamental (F20) mode PWD. The ability to suppress tissue signals using SH signals may enable the use of higher frame rates and improve sensitivity to microvascular flow or slow velocities near large vessel walls by reducing or eliminating the need for clutter filters.

In vitro characterization of the subharmonic ultrasound signal from Definity microbubbles at high frequencies.

Ultrasound microbubble contrast agents have been demonstrated to scatter subharmonic energy at one-half the driving frequency. At ultrasound frequencies in the 20-40 MHz range, the subharmonic offers the potential to differentiate the blood in the microcirculation from the surrounding tissue. It is unknown whether current contrast agents, manufactured to be resonant between 2 and 12 MHz, are ideal for subharmonic imaging at higher frequencies. We performed numerical simulations of the Keller-Miksis model for the behavior of a single bubble and experimental investigations of Definity microbubbles in water. The results supported the hypothesis that off-resonant bubbles, excited at their second harmonic, may be primarily responsible for the observed subharmonic energy. For frequencies between 20 and 32 MHz and 32 and 40 MHz, the optimal bubble diameters for the generation of subharmonics in vitro were determined experimentally to be 1.2-5 microm and less than 1.2 microm, respectively. Definity may be a suitable ultrasound contrast agent for subharmonic imaging at 20 MHz with peak-negative pressures between 380 and 590 kPa and pulses greater than or equal to four cycles in duration.

Model for the ultrasound reflection from micro-beads and cells distributed in layers on a uniform surface.

A model predicting the reflection of ultrasound from multiple layers of small scattering spheres is developed. Predictions of the reflection coefficient, which takes into account the interferences between the different sphere layers, are compared to measurements performed in the 10-80 MHz and 15-35 MHz frequency range with layers of glass beads and spherical acute myeloid leukemia (AML) cells, respectively. For both types of scatterers, the reflection coefficient increases as a function of their density on the surface for less than three superimposed layers, at which point it saturates at 0.38 for glass beads and 0.02 for AML cells. Above three layers, oscillations of the reflection coefficient due to constructive or destructive interference between layers are observed experimentally and are accurately predicted by the model. The use of such a model could lead to a better understanding of the structures observed in layered tissue images.

Estimation methods for flow imaging with high frequency ultrasound.

This article proposes to estimate slow blood flow with high frequency ultrasound imaging. The proposed technique combines 2 methods. First, a statistical method, called Speckle Flow Imaging (SFI) based on the analysis of changes in the speckle pattern along time, gives an index directly related to the total velocity vector. Secondly, a block matching approach estimates the in-plane velocity components. Results on calibrated flow sequences of blood mimicking fluid have shown good agreement with the statistical model. The quantification of flow is achieved with pulsed flow and is also angle independent when the flow is perpendicular to the ultrasound beam. Speckle Tracking has been evaluated on the same data and has shown good estimation of the in-plane velocity vector when the component of velocity perpendicular to the imaging plane is inferior to 1mm/s. The results of these two methods permit the evaluation of the total 3D velocity field and the orthogonal velocity component relative to the imaging plane. This allows the quantification of blood flow (volumetric per time unit across the sequence).

Design and fabrication of ultrafine piezoelectric composites.

Making fine scale (< 20 microm) piezoelectric composites for high frequency (> 50 MHz) ultrasound transducers remains challenging. Interdigital phase bonding (IPhB), described in this paper, presents a new technique developed to make piezoelectric composites at the ultrafine scale using a conventional dicing saw. Using the IPhB technique, a composite structure with a pitch that is less than the dicing saw blade thickness can be created. The approach is flexible enough to make composites of different combination of pitch and volume ratio. Using a conventional dicing saw with a 50 microm thick blade, composite with a 25 microm pitch and a volume ratio of 61 percent are fabricated. Such a composite is suitable for fabrication of ultrasonic transducers and arrays with central frequencies of up to 85 MHz. Single element transducers working at central frequencies of 50-60 MHz were made of these composites as a mean to characterize the acoustic performance. Measurement results of the transducers show that the longitudinal electromechanical coupling coefficient is greater than 0.6 and that there are no noticeable lateral resonances in the frequency range of 55-150 MHz. Design criteria for fine scale elements are also discussed based on theoretical results from finite element analysis (FEA).

Ultrasonic detection and developmental changes in calcification of the placenta during normal pregnancy in mice.

High resolution ultrasound imaging of the mouse placenta during development revealed highly echogenic foci localized near the materno-placental interface in early gestation and, near term, in the placental labyrinth (the exchange region of the placenta). Echogenic foci and calcium deposits identified in histological sections using Alizarin red staining showed similar localization and changes with gestation. Calcium deposits caused the echogenic foci because incubating uteri in a decalcifying solution eliminated both the deposits and echogenic foci. Transmission electron microscopy, X-ray microanalysis, and electron diffraction were used to show that deposits were calcium hydroxyapatite crystals. Calcium deposits were extensive and densely packed at days 7.5-9.5 of gestation at the border between the maternal decidua and the fetal trophoblast giant cells of ectoplacental cone. After the formation of the chorio-allantoic placenta (approximately day 10.5), calcification deposits appeared larger and more rarefied but were still localized at the border between the maternal decidua and the fetal trophoblast giant cells of the placenta. Calcification deposits were not observed in the labyrinthine region of the mouse placenta until > or = day 15.5 (day 18.5 is full term). We conclude that deposits of calcium hydroxyapatite crystals in the mouse placenta are detectable by high resolution ultrasound imaging. These deposits provide an ultrasound detectable marker of the maternal-placental interface that is particularly prominent during the establishment of the chorio-allantoic placenta between days 7.5 and 9.5 of gestation.

A new ultrasound instrument for in vivo microimaging of mice.

We report here on the design and evaluation of the first high-frequency ultrasound (US) imaging system specifically designed for microimaging of the mouse. High-frequency US or US biomicroscopy (UBM) has the advantage of low cost, rapid imaging speed, portability and high resolution. In combination with the ability to provide functional information on blood flow, UBM provides a powerful method for the investigation of development and disease models. The new UBM imaging system is demonstrated for mouse development from day 5.5 of embryogenesis through to the adult mouse. At a frequency of 40 MHz, the resolution voxel of the new mouse scanner measures 57 microm x 57 microm x 40 microm. Duplex Doppler provides blood velocity sensitivity to the mm per s range, consistent with flow in the microcirculation, and can readily detect blood flow in the embryonic mouse heart, aorta, liver and placenta. Noninvasive UBM assessment of development shows striking similarity to invasive atlases of mouse anatomy. The most detailed noninvasive in vivo images of mouse embryonic development achieved using any imaging method are presented.

Applications for multifrequency ultrasound biomicroscopy in mice from implantation to adulthood.

A new multifrequency (19-55 MHz) ultrasound biomicroscope with two-dimensional imaging and integrated Doppler ultrasound was evaluated using phantoms and isoflurane-anesthetized mice. Phantoms revealed the biomicroscope's lateral resolution was between 50 and 100 microm, whereas that of a conventional 13 MHz ultrasound system was 200-500 microm. This difference was apparent in the markedly higher resolution images achieved using the biomicroscope in vivo. Transcutaneous images of embryos in pregnant mice from approximately 2 days after implantation (7 days gestation) to near term (17.5 days) were obtained using frequencies from 25 to 40 MHz. The ectoplacental cone and early embryonic cavities were visible as were the placenta and embryonic organs throughout development to term. We also evaluated the ability of the biomicroscope to detect important features of heart development by examining embryos from 8.5 to 17.5 day gestation in exteriorized uteri using 55 MHz ultrasound. Cardiac looping, division of the outflow tract, and ventricular septation were visible. In postnatal imaging, we observed the heart and kidney of neonatal mice at 55 MHz, the carotid artery in juveniles (approximately 8 g body wt) and adults (approximately 25 g body wt) at 40 MHz, and the adult heart, aorta, and kidney at 19 MHz. The coefficient of variation of carotid and aortic diameter measurements was 1-3%. In addition, blisters in GRIP1 -/- embryos and aortic valvular stenosis in two adults were readily visualized. Using image-guided Doppler function, low blood velocities in vessels as small as 100 microm in diameter including the primitive heart tube at day 8.5 were measurable, but high blood velocities (>37.5 cm/s) such as in the heart and large arteries in late gestation and postnatal life were off-scale. Accurate cardiac dimension measurements were impeded by poor temporal resolution (4 frames/s). In summary, the multifrequency ultrasound biomicroscope is a versatile tool well suited to detailed study of the morphology of various organ systems throughout development in mice and for hemodynamic measurements in the low velocity range.

Measurement of the ultrasonic properties of human coronary arteries in vitro with a 50-MHz acoustic microscope.

Ultrasonic attenuation coefficient, wave propagation speed and integrated backscatter coefficient (IBC) of human coronary arteries were measured in vitro over the -6 dB frequency bandwidth (36 to 67 MHz) of a focused ultrasound transducer (50 MHz, focal distance 5.7 mm, f/number 1.7). Corrections were made for diffraction effects. Normal and diseased coronary artery sub-samples (N = 38) were obtained from 10 individuals at autopsy. The measured mean +/- SD of the wave speed (average over the entire vessel wall thickness) was 1581.04 +/- 53.88 m/s. At 50 MHz, the average attenuation coefficient was 4.99 +/- 1.33 dB/mm with a frequency dependence term of 1.55 +/- 0.18 determined over the 36- to 67-MHz frequency range. The IBC values were: 17.42 +/- 13.02 (sr.m)-1 for thickened intima, 11.35 +/- 6.54 (sr.m)-1 for fibrotic intima, 39.93 +/- 50.95 (sr.m)-1 for plaque, 4.26 +/- 2.34 (sr.m)-1 for foam cells, 5.12 +/- 5.85 (sr.m)-1 for media and 21.26 +/- 31.77 (sr.m)-1 for adventitia layers. The IBC results indicate the possibility for ultrasound characterization of human coronary artery wall tissue layer, including the situations of diseased arteries with the presence of thickened intima, fibrotic intima and plaque. The mean IBC normalized with respect to the mean IBC of the media layer seems promising for use as a parameter to differentiate a plaque or a thickened intima from a fibrotic intima.

Measurement of the ultrasonic properties of human coronary arteries in vitro with a 50-MHz acoustic microscope

Ultrasonic attenuation coefficient, wave propagation speed and integrated backscatter coefficient (IBC) of human coronary arteries were measured in vitro over the -6 dB frequency bandwidth (36 to 67 MHz) of a focused ultrasound transducer (50 MHz, focal distance 5.7 mm, f/number 1.7). Corrections were made for diffraction effects. Normal and diseased coronary artery sub-samples (N = 38) were obtained from 10 individuals at autopsy. The measured mean ± SD of the wave speed (average over the entire vessel wall thickness) was 1581.04 ± 53.88 m/s. At 50 MHz, the average attenuation coefficient was 4.99 ± 1.33 dB/mm with a frequency dependence term of 1.55 ± 0.18 determined over the 36- to 67-MHz frequency range. The IBC values were: 17.42 ± 13.02 (sr.m)-1 for thickened intima, 11.35 ± 6.54 (sr.m)-1 for fibrotic intima, 39.93 ± 50.95 (sr.m)-1 for plaque, 4.26 ± 2.34 (sr.m)-1 for foam cells, 5.12 ± 5.85 (sr.m)-1 for media and 21.26 ± 31.77 (sr.m)-1 for adventitia layers. The IBC results indicate the possibility for ultrasound characterization of human coronary artery wall tissue layer, including the situations of diseased arteries with the presence of thickened intima, fibrotic intima and plaque. The mean IBC normalized with respect to the mean IBC of the media layer seems promising for use as a parameter to differentiate a plaque or a thickened intima from a fibrotic intima

Ultrasonic integrated backscatter coefficient profiling of human coronary arteries in vitro.

A theoretical formulation for the profile of the integrated backscatter coefficient (IBC) is derived. This new formulation is based on a theoretical treatment by Chen et al. [1]. It includes correction for the diffraction of the ultrasonic beam and correction for the non-ideal nature of the reference signal. The inclusion of these correction factors permits accurate quantitative profiling of the IBC over the transducer focal zone. Experimental measurements are first performed on well-calibrated vessel-equivalent phantom materials and subsequently on human coronary arteries in vitro. A spherically focused 50.0 MHz f/1.83 transducer is used. IBC profiles are shown for three samples that are representative of early, mid, and advanced atherosclerotic coronary disease. The IBC profiles clearly differentiate the arterial tissues. However, variation between samples with histologically confirmed intimal thickening (N = 24) was large. The mean IBC (+/- 1 standard deviation), in (Sr.mm)-1, for media, adventitia, and thickened intima were 3.86 x 10(-3), 1.53 x 10(-2), and 2.24 x 10(-2), respectively. The mean IBC of thickened intima is larger than previous measurements obtained from femoral arteries, and the mean IBC for media and adventitia layers are lower, reflecting differences in tissue composition between coronary and femoral vessels.

Interdigital pair bonding for high frequency (20-50 MHz) ultrasonic composite transducers.

Interdigital pair bonding is a novel methodology that enables the fabrication of high frequency piezoelectric composites with high volume fractions of the ceramic phase. This enhancement in ceramic volume fraction significantly reduces the dimensional scale of the epoxy phase and increases the related effective physical parameters of the composite, such as dielectric constant and the longitudinal sound velocity, which are major concerns in the development of high frequency piezoelectric composites. In this paper, a method called interdigital pair bonding (IPB) is used to prepare 1-3 piezoelectric composite with a pitch of 40 microns, a kerf of 4 microns, and a ceramic volume fraction of 81%. The composites prepared in this fashion exhibited a very pure thickness-mode resonance up to a frequency of 50 MHz. Unlike the 2-2 piezoelectric composites with the same ceramic and epoxy scales developed earlier, the anticipated lateral modes between 50 to 100 MHz were not observed in the current 1-3 composites. The mechanisms for the elimination of the lateral modes at high frequency are discussed. The effective electromechanical coupling coefficient of the composite was 0.72 at a frequency of 50 MHz. The composites showed a high longitudinal sound velocity of 4300 m/s and a high clamped dielectric constant of 1111 epsilon 0, which will benefit the development of high frequency ultrasonic transducers and especially high frequency transducer arrays for medical imaging.

In vitro and in vivo comparison of three different intravascular ultrasound catheter designs.

Intravascular ultrasound (IVUS) is an invasive imaging modality, which provides detailed two-dimensional images of blood vessels. There are currently two different types of IVUS catheters available, namely, the phased-array and the mechanical designs. The operating ultrasound frequency of these catheters ranges from 20 to 40 MHz. This study sought to evaluate the image quality, accuracy of diameter and pullback length measurements, and catheter handling characteristics of three different IVUS catheters currently available for clinical use using both in vitro phantom models and in patients undergoing percutaneous coronary intervention (PCI). In gelatin phantom models, image quality assessed on a semiquantitative scale was significantly different between the three IVUS catheters (P = 0.01) with the 40-MHz catheter providing the best images. Accuracy of lumen diameter measurements, when compared to optical microscopy, were similar between the three IVUS catheter designs (all R(2) = 0.99). There were no significant differences in accuracy of pullback length measurements in vitro between the three designs. However, there were differences in the performance of the three IVUS catheters when used for preinterventional imaging in patients undergoing PCI. Both mechanical IVUS catheters were associated with lower procedural, fluoroscopy, and lesion crossing times compared to the phased-array catheter (all P < 0.05). There were no significant differences between the catheters during postinterventional IVUS imaging. There were also small but potentially important differences with regards to clinical events and complications associated with the use of the different IVUS catheters during the PCI procedures, reflecting differences in catheter design.

Interstitial ultrasound heating applicator for MR-guided thermal therapy.

The ability to control the shape of thermal coagulation was investigated for various interstitial heating applicators incorporating planar transducers and device rotation. Magnetic-resonance-compatible interstitial ultrasound applicators were constructed and the effects of ultrasound power, frequency, scan rate and heating time on lesion radius were studied in heating experiments in excised liver tissue. Continuous thermal lesions were generated by scanning heating applicators over a 180 angular sector. The region of thermal coagulation was restricted to the prescribed sector. Lesion radius increased with acoustic power and heating time and decreased with increasing frequency. The relationship between the temperature distribution generated by the applicator and the resulting thermal lesion was assessed with MRI. Analysis of MR temperature maps revealed that the temperature distribution could be measured accurately within 2 mm from the surface of the applicator, and the boundary of thermal coagulation was defined by a temperature of 54 +/- 12 degrees C. Calculations of temperature distributions indicated that slower scan rates can overcome the tendency of perfusion to reduce the radius of thermal lesion. This applicator design and delivery strategy make conformal interstitial heating possible.

High frequency acoustic parameters of human and bovine articular cartilage following experimentally-induced matrix degradation.

Matrix degradation and proteoglycan loss in articular cartilag eare features of early osteoarthritis. To determine the effect of matrix degradation and proteoglycan loss on ultrasound propagation in cartilage, we used papain and interleukin-1alpha to degrade the matrix proteoglycans of human and bovine cartilage samples, respectively. There is also minor collagen alteration associated with these chemical degradation methods. We compared the speed of sound and frequency dependent attenuation (20-40 MHz) of control and experimental paired samples. We found that a loss of matrix proteoglycans and collagen disruption resulted in a 20-30% increase in the frequency dependent attenuation and a 2% decrease in the speed of sound in both human and bovine cartilage. We conclude that the frequency dependent attenuation and speed of sound in articular cartilage are sensitive to experimental modification of the matrix proteoglycans and collagen. These findings suggest that ultrasound can potentially be used to detect morphologic changes in articular cartilage associated with the progression of osteoarthritis.

Feasibility of linear arrays for interstitial ultrasound thermal therapy.

The feasibility of linear array transducers for interstitial ultrasound thermal therapy was evaluated. Theoretical acoustic power distributions were used to calculate spatial heating patterns using the bioheat transfer equation. The spatial heating patterns of linear array and single element planar rectangular transducers were compared. Scanned heating with both transducer geometries produced asymmetric heating volumes; however, a more uniform radial temperature profile with a sharper margin was achieved with linear arrays. Single element transducers produced excessive heating near the probe surface. Homogeneous blood flow is predicted to reduce the mean temperature within the heated region, with little effect on the spatial pattern.