High-Frequency Ex vivo Ultrasound Imaging of the Auditory System

A 50 MHz array-based imaging system was used to obtain high-resolution images of the ear and auditory system. This previously described custom built imaging system (3, 4 and 2) is capable of 50 μm axial resolution, and lateral resolution varying from 80 μm to 130 μm over a 5.12 mm scan depth. The imaging system is based on a 2 mm diameter, seven-element equal-area annular array, and a digital beamformer that uses high-speed field programmable gate arrays (FPGAs). The images produced by this system have shown far superior depth of field compared with commercially available single-element systems. Ex vivo, three-dimensional (3-D) images were obtained of human cadaveric tissues including the ossicles (stapes, incus, malleus) and the tympanic membrane. In addition, two-dimensional (2-D) images were obtained of an intact cochlea by imaging through the round window membrane. The basilar membrane inside the cochlea could clearly be visualized. These images demonstrate that high-frequency ultrasound imaging of the middle and inner ear can provide valuable diagnostic information using minimally invasive techniques that could potentially be implemented in vivo. (E-mail: J.Brown@dal.ca)     

Contrast-Enhanced Intravascular Ultrasound Pulse Sequences for Bandwidth-Limited Transducers

We demonstrate two methods for vasa vasorum imaging using contrast-enhanced intravascular ultrasound, which can be performed using commercial catheters. Plaque neovascularization was recognized as an independent marker of coronary artery plaque vulnerability. IVUS-based methods to image the microvessels available to date require high bandwidth (−6 dB relative frequency bandwidth >70%), which are not routinely available commercially. We explored the potential of ultraharmonic imaging and chirp reversal imaging for vasa vasorum imaging. In vitro recordings were performed on a tissue-mimicking phantom using a commercial ultrasound contrast agent and a transducer with a center frequency of 34 MHz and a −6 dB relative bandwidth of 56%. Acoustic peak pressures <500 kPa were used. A tissue-mimicking phantom with channels down to 200 μm in diameter was successfully imaged by the two contrast detection sequences while the smallest channel stayed invisible in conventional intravascular ultrasound images. Ultraharmonic imaging provided the best contrast agent detection.     

Ultrasound Phase Contrast Thermal Imaging with Reflex Transmission Imaging Methods in Tissue Phantoms

Thermal imaging measurements using ultrasound phase contrast have been performed in tissue phantoms heated with a focused ultrasound source. Back projection and reflex transmission imaging principles were used to detect sound speed–induced changes in the phase caused by an increase in the temperature. The temperature was determined from an empirical relationship for the temperature dependence on sound speed. The phase contrast was determined from changes in the sound field measured with a hydrophone scan conducted before and during applied heating. The lengthy scanning routine used to mimic a large two-dimensional array required a steady-state temperature distribution within the phantom. The temperature distribution in the phantom was validated with magnetic resonance (MR) thermal imaging measurements. The peak temperature was found to agree within 1°C with MR, and good agreement was found between the temperature profiles. The spatial resolution was 0.3 × 0.3 × 0.3 mm, comparing favorably with the 0.625 × 0.625 × 1.5-mm MR spatial resolution. (E-mail: cfarny@bwh.harvard.edu)     

A New 15–50 MHz Array-Based Micro-Ultrasound Scanner for Preclinical Imaging

Most institutions now have a suite of imaging tools to follow mouse models of human disease. Micro-ultrasound is one of these tools and is second after whole-mouse fluorescence or bioluminescent imaging, in terms of installed systems. We report in this paper the first commercially available array transducer–based ultrasound imaging system that enables micro-ultrasound imaging at center frequencies between 15 and 50 MHz. At the heart of the new scanner is a laser-machined high-frequency 256 element, linear transducer array capable of forming dynamic diffraction limited beams. The power of the linear array approach is embodied in the uniform high resolution maintained over the full field of view. This leads to greatly expanded scope for real-time functional imaging that is demonstrated in this paper. The unprecedented images made with the new imaging system will enable many new applications not previously possible. These include real-time visualization of flow in the mouse placenta, visualization of flow development in the embryo, studies of embryonic to adult cardiac development/disease, and studies of real-time blood flow in mouse models of tumour angiogenesis. (E-mail: Stuart.foster@sunnybrook.ca)     

Blood-Brain Barrier (BBB) Disruption Using a Diagnostic Ultrasound Scanner and Definity® in Mice

The objective of this work was to determine whether diagnostic ultrasound and contrast agent could be used to transcranially and nondestructively disrupt the blood-brain barrier (BBB) in mice under ultrasound image guidance and to quantify that disruption using magnetic resonance imaging (MRI) and magnetic resonance (MR) contrast agent. Each mouse was placed under isoflurane anesthesia and the hair on top of its skull was removed before treatment. A diagnostic ultrasound transducer was placed in a water bag coupled with gel on the mouse skull. Definity (ultrasound [US] contrast) and Magnevist (MR contrast) were injected concurrent with the start of a custom ultrasound transmission sequence. The transducer was translated along the rostral-caudal axis to insonify three spatial locations (2 mm apart) along one half of the brain for each sequence. T1-weighted MR images were used to quantify the volume of tissue over which the BBB disruption allowed Magnevist to enter the brain, based upon increases in MR contrast-to-noise ratio (CNR) compared with the noninsonified portions of the brain. Ultrasonic frequency, pressure and pulse duration, as well as Definity dose and injection time were varied. Preliminary results suggest that a threshold exists for BBB opening dependent upon both pressure and pulse duration (consistent with reports in the literature performed at lower frequencies). A range of typical diagnostic frequencies (e.g., 5.0–8.0 MHz) generated BBB disruption. Comparable BBB opening was noted with varied delays between Definity injection and insonification (0–2 min) for a range of Definity concentrations (400-2400 μL/kg). The low-pressure, custom sequences (mechanical index [MI] ≤ 0.65) had minimal blood cell extravasation as determined by histologic evaluation. This study has shown the ability of a diagnostic ultrasound system, in conjunction with Definity, to open the BBB transcranially in a mouse model for molecules approximately 0.5 kDa in size. Opening was achieved at higher frequencies than previously reported and was localized under ultrasound image guidance. A typical, ultrasound imaging mode (pulsed wave [PW] Doppler) with specific settings (transmit frequency = 5.7 MHz, gate size = 15 mm, pulse repetition frequency = 100 Hz, system power = 15%) successfully opened the BBB, which facilitates implementation using the most of commercially available clinical diagnostic scanners. Localized opening of the BBB may have potential clinical utility for the delivery of diagnostic or therapeutic agents to the brain. (Email: kdf2@duke.edu)     

Ultrasound Backscatter Imaging Provides Frequency-Dependent Information on Structure,Composition and Mechanical Properties of Human Trabecular Bone

The strength as well as the acoustic properties of trabecular bone are determined by its structure and composition. Consequently, tissue structure and compositional properties also affect the ultrasound propagation in bone. The diagnostic potential of ultrasound has not been fully exploited in clinical quantitative ultrasound devices. The aim of this study was to investigate the ability of quantitative ultrasound pulse-echo imaging, conducted over a broad range of frequencies (1 to 5 MHz), to predict the mechanics, composition and microstructure of trabecular bone. Ultrasound reflection and backscatter parameters correlated significantly with the ultimate strength of the trabecular bone and the bone volume fraction (r = 0.76–0.90, n = 20, p < 0.01). Ultrasound backscatter associated significantly (independently of bone structure or mineral content) with the collagen content of the bone matrix (r = 0.75, r_adjusted = 0.66, p < 0.01). Interestingly, the applied ultrasound frequency seemed to relate the sensitivity of ultrasound backscatter to different properties of trabecular bone. At frequencies ranging from 1 to 3.5 MHz, the ultrasound backscatter associated significantly with the tissue mechanical and structural parameters. At 5 MHz, the composition of the bone matrix was a more significant determinant of the measured backscatter. This study provides useful information for optimizing the use of pulse-echo measurements, and thereby further emphasizes the diagnostic potential of the ultrasound backscatter measurements of trabecular bone.     

40-MHz annular array imaging of mouse embryos

Ultrasound biomicroscopy (UBM) has emerged as an important in vivo imaging approach for analyzing normal and genetically engineered mouse embryos. Current UBM systems use fixed-focus transducers, which are limited in depth-of-focus. Depending on the gestational age of the embryo, regions-of-interest in the image can extend well beyond the depth-of-focus for a fixed-focus transducer. This shortcoming makes it particularly problematic to analyze 3-D data sets and to generate accurate volumetric renderings of the mouse embryonic anatomy. To address this problem, we have developed a five-element, 40-MHz annular array transducer and a computer-controlled system to acquire and reconstruct fixed- and array-focused images of mouse embryos. Both qualitative and quantitative comparisons showed significant improvement with array-focusing, including an increase of 3 to 9 dB in signal-to-noise ratio and an increase of at least 2.5 mm in depth-of-focus. Volumetric-rendered images of brain ventricles demonstrated the clear superiority of array-focusing for 3-D analysis of mouse embryonic anatomy.     

Longitudinal in Utero Analysis of Engrailed-1 Knockout Mouse Embryonic Phenotypes Using High-Frequency Ultrasound

Large-scale international efforts to generate and analyze loss-of-function mutations in each of the approximately 20,000 protein-encoding gene mutations are ongoing using the “knockout” mouse as a model organism. Because one-third of gene knockouts are expected to result in embryonic lethality, it is important to develop non-invasive in utero imaging methods to detect and monitor mutant phenotypes in mouse embryos. We describe the utility of 3-D high-frequency (40-MHz) ultrasound (HFU) for longitudinal in utero imaging of mouse embryos between embryonic days (E) 11.5 and E14.5, which represent critical stages of brain and organ development. Engrailed-1 knockout (En1-ko) mouse embryos and their normal control littermates were imaged with HFU in 3-D, enabling visualization of morphological phenotypes in the developing brains, limbs and heads of the En1-ko embryos. Recently developed deep learning approaches were used to automatically segment the embryonic brain ventricles and bodies from the 3-D HFU images, allowing quantitative volumetric analyses of the En1-ko brain phenotypes. Taken together, these results show great promise for the application of longitudinal 3-D HFU to analyze knockout mouse embryos in utero.     

In vivo wide-field reflectance/fluorescence imaging and polarization-sensitive optical coherence tomography of human oral cavity with a forward-viewing probe

We report multimodal imaging of human oral cavity in vivo based on simultaneous wide-field reflectance/fluorescence imaging and polarization-sensitive optical coherence tomography (PS-OCT) with a forward-viewing imaging probe. Wide-field reflectance/fluorescence imaging and PS-OCT were to provide both morphological and fluorescence information on the surface, and structural and birefringent information below the surface respectively. The forward-viewing probe was designed to access the oral cavity through the mouth with dimensions of approximately 10 mm in diameter and 180 mm in length. The probe had field of view (FOV) of approximately 5.5 mm in diameter, and adjustable depth of field (DOF) from 2 mm to 10 mm by controlling numerical aperture (NA) in the detection path. This adjustable DOF was to accommodate both requirements for image-based guiding with high DOF and high-resolution, high-sensitivity imaging with low DOF. This multimodal imaging system was characterized by using a tissue phantom and a mouse model in vivo, and was applied to human oral cavity. Information of surface morphology and vasculature, and under-surface layered structure and birefringence of the oral cavity tissues was obtained. These results showed feasibility of this multimodal imaging system as a tool for studying oral cavity lesions in clinical applications.OCIS codes: (170.4500) Optical coherence tomography, (300.2530) Fluorescence, laser-induced, (170.3880) Medical and biological imaging, (170.3890) Medical optics instrumentation     

Ultrafast Imaging of Ultrasound Contrast Agents

The disappearance of ultrasound contrast agents after disruption can provide useful information on their environment. However, in vivo acoustical imaging of this transient phenomenon, which has a duration on the order of milliseconds, requires high frame rates that are unattainable by conventional ultrasound scanners. In this article, ultrafast imaging is applied to microbubble tracking using a 128-element linear array and an elastography scanner. Contrast agents flowing in a wall-less tissue phantom are insonified with a high-intensity disruption pulse followed by a series of plane waves emitted at a 5 kHz PRF. A collection of compounded images depicting the evolution of microbubbles is obtained after the echoes are beamformed in silico. The backscattering of the microbubbles appears to increase in the first image after disruption (4 ms) and decrease following an exponential decay in the next hundred milliseconds. This microbubble dynamic depends on the length and amplitude of the high-intensity pulse. Furthermore, confined microbubbles are found to differ significantly from their free-flowing counterparts in their dissolution curves. The high temporal resolution provided by ultrafast imaging could help distinguish targeted microbubbles during molecular imaging. (E-mail: olicou@gmail.com)     

7.5 MHz dual-layer transducer array for 3-D rectilinear imaging

The difficulties associated with fabrication and interconnection have limited the development of 2-D ultrasound transducer arrays with a large number ofelements (>5000). In previous work, we described a 5 MHz center frequency PZT-P[VDF-TrFE] dual-layer transducer that used two perpendicular 1-D arrays for 3-D rectilinear imaging. This design substantially reduces the channel count as well as fabrication complexity, which makes 3-D imaging more realizable. Higher frequencies (>5 MHz) are more commonly used in clinical applications or imaging targets near transducers, such as the breast, carotid and musculoskeletal tissue. In this paper, we present a 7.5 MHz dual-layer transducer array for 3-D rectilinear imaging. A modified acoustic stack model was designed and fabricated. PZT elements were sub-diced to eliminate lateral coupling. This sub-dicing process made the PZT into a 2-2 composite material, which could help improve transducer sensitivity and bandwidth. Full synthetic-aperture 3-D data sets were acquired by interfacing the transducer with a Verasonics data-acquisition system (VDAS). Offline 3-D beamforming was then performed to obtain volumes of a multiwire phantom and a cyst phantom. The generalized coherence factor (GCF) was applied to improve the contrast of cyst images. The measured -6 dB fractional bandwidth of the transducer was 71% with a center frequency of 7.5 MHz. The measured lateral beamwidths were 0.521 mm and 0.482 mm in azimuth and elevation, respectively, compared with a simulated beamwidth of 0.43 mm.     

3-D-Printed Registration Phantom for Combined Ultrasound and Optical Imaging of Biological Tissues

Efforts to develop quantitative ultrasound biomarkers would benefit from comparisons between ultrasound data and higher-resolution images of the tissue microstructure, such as from optical microscopy. However, only a few studies have used these methods for multiscale imaging because it is difficult to register low-resolution (>100 μm) ultrasound images to high-resolution microscopy images. To address this need, we have designed a 3-D-printed registration phantom that is made of a hard fluorescent resin, fits into a glass-bottom dish and can be used to calculate a coordinate system transform between ultrasound and optical microscopy. We report the phantom design, a registration protocol and an example registration using 18.5-MHz ultrasound and second harmonic generation microscopy. We evaluate the registration precision, achieving standard deviations smaller than the ultrasound resolution across all axes, and illustrate on a mouse mammary gland that this method yields results superior to those of manual landmark registration.     

High-Frequency Micro-Ultrasound Imaging and Optical Topographic Imaging for Spinal Surgery: Initial Experiences

High frequency micro-ultrasound (µUS) transducers with central frequencies up to 50?MHz facilitate dynamic visualization of patient anatomy with minimal disruption of the surgical work flow. Micro-ultrasound improves spatial resolution over conventional ultrasound imaging from millimeter to micrometer, but compromises depth penetration. This trade-off is sufficient during an open surgery in which the bone is removed and theultrasound probe can be placed into the surgical cavity. By fusing µUS with pre-operative imaging and tracking the ultrasound probe intra-operatively using our optical topographic imaging technology, we can provide dynamic feedback during surgery, thus affecting clinical decision making. We present our initial experience using high-frequency µUS imaging during spinal procedures. Micro-ultrasound images were obtained in five spinal procedures. Medical rationale for use of µUS was provided for each patient. Surgical procedures were performed using the standard clinical practice with bone removal to facilitate real-time ultrasound imaging of the soft tissue. During surgery, the µUS probe was registered to the pre-operative computed tomography and magnetic resonance images. Images obtained comprised five spinal decompression surgeries (four tumor resections, one cystic synovial mass). Micro-ultrasound images obtained during spine surgery delineated exquisite detailing of the spinal anatomy including white matter and gray matter tracts and nerve roots and allowed accurate assessment of the extent of decompression/tumor resection. In conclusion, tracked µUS enables real-time imaging of the surgical cavity, conferring significant qualitative improvement over conventional ultrasound.     

Very Low Frequency Radial Modulation for Deep Penetration Contrast-Enhanced Ultrasound Imaging

Contrast-enhanced ultrasound imaging allows vascular imaging in a variety of diseases. Radial modulation imaging is a contrast agent-specific imaging approach for improving microbubble detection at high imaging frequencies (≥7.5 MHz), with imaging depth limited to a few centimeters. To provide high-sensitivity contrast-enhanced ultrasound imaging at high penetration depths, a new radial modulation imaging strategy using a very low frequency (100 kHz) ultrasound modulation wave in combination with imaging pulses ≤5 MHz is proposed. Microbubbles driven at 100 kHz were imaged in 10 successive oscillation states by manipulating the pulse repetition frequency to unlock the frame rate from the number of oscillation states. Tissue background was suppressed using frequency domain radial modulation imaging (F-RMI) and singular value decomposition-based radial modulation imaging (S-RMI). One hundred-kilohertz modulation resulted in significantly higher microbubble signal magnitude (63–88 dB) at the modulation frequency relative to that without 100-kHz modulation (51–59 dB). F-RMI produced images with high contrast-to-tissue ratios (CTRs) of 15 to 22 dB in a stationary tissue phantom, while S-RMI further improved the CTR (19–26 dB). These CTR values were significantly higher than that of amplitude modulation pulse inversion images (11.9 dB). In the presence of tissue motion (1 and 10 mm/s), S-RMI produced high-contrast images with CTR up to 18 dB; however, F-RMI resulted in minimal contrast enhancement in the presence of tissue motion. Finally, in transcranial ultrasound imaging studies through a highly attenuating ex vivo cranial bone, CTR values with S-RMI were as high as 23 dB. The proposed technique demonstrates successful modulation of microbubble response at 100 kHz for the first time. The presented S-RMI low-frequency radial modulation imaging strategy represents the first demonstration of real-time (20 frames/s), high-penetration-depth radial modulation imaging for contrast-enhanced ultrasound imaging.     

Registration of 3D trans-esophageal echocardiography to X-ray fluoroscopy using image-based probe tracking

Two-dimensional (2D) X-ray imaging is the dominant imaging modality for cardiac interventions. However, the use of X-ray fluoroscopy alone is inadequate for the guidance of procedures that require soft-tissue information, for example, the treatment of structural heart disease. The recent availability of three-dimensional (3D) trans-esophageal echocardiography (TEE) provides cardiologists with real-time 3D imaging of cardiac anatomy. Increasingly X-ray imaging is now supported by using intra-procedure 3D TEE imaging. We hypothesize that the real-time co-registration and visualization of 3D TEE and X-ray fluoroscopy data will provide a powerful guidance tool for cardiologists. In this paper, we propose a novel, robust and efficient method for performing this registration. The major advantage of our method is that it does not rely on any additional tracking hardware and therefore can be deployed straightforwardly into any interventional laboratory. Our method consists of an image-based TEE probe localization algorithm and a calibration procedure. While the calibration needs to be done only once, the GPU-accelerated registration takes approximately from 2 to 15 s to complete depending on the number of X-ray images used in the registration and the image resolution. The accuracy of our method was assessed using a realistic heart phantom. The target registration error (TRE) for the heart phantom was less than 2 mm. In addition, we assess the accuracy and the clinical feasibility of our method using five patient datasets, two of which were acquired from cardiac electrophysiology procedures and three from trans-catheter aortic valve implantation procedures. The registration results showed our technique had mean registration errors of 1.5-4.2 mm and 95% capture range of 8.7-11.4 mm in terms of TRE.     

Effects of phase aberration on high frame rate imaging

A high frame-rate (HFR) imaging method (about 3750 frames/s for imaging of biological soft tissues at a depth of 200 mm) has been developed recently with limited diffraction beams. This method uses the fast Fourier transform (FFT) and inverse fast Fourier transform (IFFT) to construct images, and can be implemented with simple and inexpensive hardware, compared to the conventional delay-and-sum method where a digital beam former is usually used. In this paper, phase aberration effects are studied for both the high frame rate and the conventional methods by adding random phase shifts to echo signals obtained from an experiment. In the study, two broadband linear arrays were used to construct images of an ATS 539 tissue-equivalent phantom that has a frequency-dependent attenuation of about 0.5 dB/MHz/cm. The first array has 48 elements, a central frequency of 2.25 MHz, an aperture of 18.288 mm, and a width of 12.192 mm in elevation. The second has 64 elements, a central frequency of 2.5 MHz, and a dimension of 38.4 mm x 10 mm. The-6dB pulse-echo bandwidth of both arrays is about 40% of their center frequencies. Radiofrequency (RF) signals were digitized at 20 mega samples/s at a 12-bit resolution to construct images. Results show that phase aberration has about the same effect on both methods in terms of image resolution and contrast, although the high frame-rate method can be implemented with a simpler system.     

Fast,Low-Frequency Plane-Wave Imaging for Ultrasound Contrast Imaging

Plane-wave ultrasound contrast imaging offers a faster, less destructive means for imaging microbubbles compared with traditional ultrasound imaging. Even though many of the most acoustically responsive microbubbles have resonant frequencies in the lower-megahertz range, higher frequencies (>3 MHz) have typically been employed to achieve high spatial resolution. In this work we implement and optimize low-frequency (1.5-4 MHz) plane-wave pulse inversion imaging on a commercial, phased-array imaging transducer in vitro and illustrate its use in vivo by imaging a mouse xenograft model. We found that the 1.8-MHz contrast signal was about four times that acquired at 3.1 MHz on matched probes and nine times greater than echoes received on a higher-frequency probe. Low-frequency imaging was also much more resilient to motion. In vivo, we could identify sub-millimeter vasculature inside a xenograft tumor model and easily assess microbubble half-life. Our results indicate that low-frequency imaging can provide better signal-to-noise because it generates stronger non-linear responses. Combined with high-speed plane-wave imaging, this method could open the door to super-resolution imaging at depth, while high power pulses could be used for image-guided therapeutics.     

High‐Frequency Ultrasound to Grade Disease Progression in Murine Models of Duchenne Muscular Dystrophy

Objective. This study used high‐frequency ultrasound (HFU) imaging to assess muscle damage noninvasively in a longitudinal study of 2 transgenic murine models of Duchenne muscular dystrophy (DMD): mdx, which has mutated cytoskeletal protein dystrophin; and udx, which has mutated dystrophin and lacks another cytoskeleton protein, utrophin. The mdx group was further subdivided into exercised and nonexercised subgroups to assess exercise‐induced damage. Methods. Muscle damage was assessed with HFU imaging (40 MHz) at biweekly intervals for 16 weeks. The assessment was based on the number of hyperechoic lesions, the lesion diameter, and muscle disorganization, giving a combined grade according to a 5‐point scale. Results. High‐frequency ultrasound discriminated the severity of muscle damage between wild‐type and transgenic models of DMD and between mdx and udx models. Qualitative comparisons of 3‐dimensional HFU images with serial histologic sections of the skeletal muscle showed the ability of ultrasound to accurately depict changes seen in the muscle architecture in vivo. Conclusions. High‐frequency ultrasound images soft tissue in mice at high contrast and spatial resolution, thereby showing that this microimaging modality has the capability to assess architectural changes in muscle fibers due to myotonic dystrophy–related diseases such as DMD.     

Simultaneous Bilateral Real-Time 3-D Transcranial Ultrasound Imaging at 1 MHz Through Poor Acoustic Windows

Ultrasound imaging has been proposed as a rapid, portable alternative imaging modality to examine stroke patients in pre-hospital or emergency room settings. However, in performing transcranial ultrasound examinations, 8%–29% of patients in a general population may present with window failure, in which case it is not possible to acquire clinically useful sonographic information through the temporal bone acoustic window. In this work, we describe the technical considerations, design and fabrication of low-frequency (1.2 MHz), large aperture (25.3 mm) sparse matrix array transducers for 3-D imaging in the event of window failure. These transducers are integrated into a system for real-time 3-D bilateral transcranial imaging—the ultrasound brain helmet—and color flow imaging capabilities at 1.2 MHz are directly compared with arrays operating at 1.8 MHz in a flow phantom with attenuation comparable to the in vivo case. Contrast-enhanced imaging allowed visualization of arteries of the Circle of Willis in 5 of 5 subjects and 8 of 10 sides of the head despite probe placement outside of the acoustic window. Results suggest that this type of transducer may allow acquisition of useful images either in individuals with poor windows or outside of the temporal acoustic window in the field.     

High-frequency 3-D color-flow imaging of the microcirculation

High-frequency (> 20 MHz) ultrasound (US) flow imaging has the potential to be an important tool for assessing microvascular blood flow in superficial tissues noninvasively. In this paper, we describe the development and evaluation of a 3-D US flow imaging system capable of operating at center frequencies in the 20- to 50-MHz range. Flow images are made for tissue volumes of sizes up to 10 mm laterally and 5 mm in depth, permitting a range of scientific and clinical applications. To acquire data sets in a reasonable time, the 2-D sections were derived from data collected with a transducer that was scanning continuously in a direction perpendicular to the beam axis. Due to spectral broadening effects induced by scanning tissue, significant tradeoffs must be made between frame rate, lateral resolution and the minimum detectable blood velocity. 3-D flow images were reconstructed with flow data acquired from a series of adjacent planes. The system was evaluated at a center frequency of 50 MHz, using two PVDF transducers with lateral resolutions of 43 microm and 65 microm and axial resolutions of 66 microm to 72 microm, respectively. Velocity ranges were from below 1 mm/s to 25 mm/s. In vivo validation experiments using the mouse ear demonstrated the ability to follow branching patterns of closely spaced microvessels from 30 microm to 100 microm in diameter. Experiments conducted on mouse tumors successfully imaged microvessel morphology in the tumor microcirculation.