ULTRA-SR Challenge: Assessment of Ultrasound Localization and TRacking Algorithms for Super-Resolution Imaging.

With the widespread interest and uptake of super-resolution ultrasound (SRUS) through localization and tracking of microbubbles, also known as ultrasound localization microscopy (ULM), many localization and tracking algorithms have been developed. ULM can image many centimeters into tissue in-vivo and track microvascular flow non-invasively with sub-diffraction resolution. In a significant community effort, we organized a challenge, Ultrasound Localization and TRacking Algorithms for Super-Resolution (ULTRA-SR). The aims of this paper are threefold: to describe the challenge organization, data generation, and winning algorithms; to present the metrics and methods for evaluating challenge entrants; and to report results and findings of the evaluation. Realistic ultrasound datasets containing microvascular flow for different clinical ultrasound frequencies were simulated, using vascular flow physics, acoustic field simulation and nonlinear bubble dynamics simulation. Based on these datasets, 38 submissions from 24 research groups were evaluated against ground truth using an evaluation framework with six metrics, three for localization and three for tracking. In-vivo mouse brain and human lymph node data were also provided, and performance assessed by an expert panel. Winning algorithms are described and discussed. The publicly available data with ground truth and the defined metrics for both localization and tracking present a valuable resource for researchers to benchmark algorithms and software, identify optimized methods/software for their data, and provide insight into the current limits of the field. In conclusion, Ultra-SR challenge has provided benchmarking data and tools as well as direct comparison and insights for a number of the state-of-the art localization and tracking algorithms.

Differentiation of Vascular Characteristics Using Contrast-Enhanced Ultrasound Imaging.

Ultrasound contrast imaging has been used to assess tumour growth and regression by assessing the flow through the macro- and micro-vasculature. Our aim was to differentiate the blood kinetics of vessels such as veins, arteries and microvasculature within the limits of the spatial resolution of contrast-enhanced ultrasound imaging. The highly vascularised ovine ovary was used as a biological model. Perfusion of the ovary with SonoVue was recorded with a Philips iU22 scanner in contrast imaging mode. One ewe was treated with prostaglandin to induce vascular regression. Time-intensity curves (TIC) for different regions of interest were obtained, a lognormal model was fitted and flow parameters calculated. Parametric maps of the whole imaging plane were generated for 2 × 2 pixel regions of interest. Further analysis of TICs from selected locations helped specify parameters associated with differentiation into four categories of vessels (arteries, veins, medium-sized vessels and micro-vessels). Time-dependent parameters were associated with large veins, whereas intensity-dependent parameters were associated with large arteries. Further development may enable automation of the technique as an efficient way of monitoring vessel distributions in a clinical setting using currently available scanners.

Super-Resolution Contrast-Enhanced Ultrasound Methodology for the Identification of In Vivo Vascular Dynamics in 2D.

OBJECTIVES: The aim of this study was to provide an ultrasound-based super-resolution methodology that can be implemented using clinical 2-dimensional ultrasound equipment and standard contrast-enhanced ultrasound modes. In addition, the aim is to achieve this for true-to-life patient imaging conditions, including realistic examination times of a few minutes and adequate image penetration depths that can be used to scan entire organs without sacrificing current super-resolution ultrasound imaging performance. METHODS: Standard contrast-enhanced ultrasound was used along with bolus or infusion injections of SonoVue (Bracco, Geneva, Switzerland) microbubble (MB) suspensions. An image analysis methodology, translated from light microscopy algorithms, was developed for use with ultrasound contrast imaging video data. New features that are tailored for ultrasound contrast image data were developed for MB detection and segmentation, so that the algorithm can deal with single and overlapping MBs. The method was tested initially on synthetic data, then with a simple microvessel phantom, and then with in vivo ultrasound contrast video loops from sheep ovaries. Tracks detailing the vascular structure and corresponding velocity map of the sheep ovary were reconstructed. Images acquired from light microscopy, optical projection tomography, and optical coherence tomography were compared with the vasculature network that was revealed in the ultrasound contrast data. The final method was applied to clinical prostate data as a proof of principle. RESULTS: Features of the ovary identified in optical modalities mentioned previously were also identified in the ultrasound super-resolution density maps. Follicular areas, follicle wall, vessel diameter, and tissue dimensions were very similar. An approximately 8.5-fold resolution gain was demonstrated in vessel width, as vessels of width down to 60 µm were detected and verified (λ = 514 µm). Best agreement was found between ultrasound measurements and optical coherence tomography with 10% difference in the measured vessel widths, whereas ex vivo microscopy measurements were significantly lower by 43% on average. The results were mostly achieved using video loops of under 2-minute duration that included respiratory motion. A feasibility study on a human prostate showed good agreement between density and velocity ultrasound maps with the histological evaluation of the location of a tumor. CONCLUSIONS: The feasibility of a 2-dimensional contrast-enhanced ultrasound-based super-resolution method was demonstrated using in vitro, synthetic and in vivo animal data. The method reduces the examination times to a few minutes using state-of-the-art ultrasound equipment and can provide super-resolution maps for an entire prostate with similar resolution to that achieved in other studies.

Resolving Ultrasound Contrast Microbubbles Using Minimum Variance Beamforming.

Minimum Variance (MV) beamforming is known to improve the lateral resolution of ultrasound images and enhance the separation of isolated point scatterers. This paper aims to evaluate the adaptive beamformer's performance with flowing microbubbles (MBs) which are relevant to super-resolution ultrasound imaging. Simulations using point scatterer data from single emissions were complemented by an experimental investigation performed using a capillary tube phantom and the Synthetic Aperture Real-time Ultrasound System (SARUS). The MV performance was assessed by the minimum distance that allows the display of two scatterers positioned side-by-side, the lateral Full-Width-at-Half-Maximum (FWHM), and the Peak-Sidelobe-Level (PSL). In the tube, scatterer responses separated by down to [Formula: see text] (or 1.05λ ) were distinguished by the MV method, while the standard Delay-And-Sum (DAS) beamformers were unable to achieve such separation. Up to ninefold FWHM decrease was also measured in favor of the MV beamformer for individual echoes from MBs. The lateral distance between two scatterers impacted on their FWHM value, and additional differences in the scatterers' axial or out-of-plane position also impacted on their size and appearance. The simulation and experimental results were in agreement in terms of lateral resolution. The point scatterer study showed that the proposed MV imaging scheme provided clear resolution benefits compared to DAS. Current super-resolution methods mainly depend on DAS beamformers. Instead, the use of the MV method may provide a larger number of detected, and potentially better localized, MB scatterers.

High-Frame-Rate Contrast Echocardiography Using Diverging Waves: Initial In Vitro and In Vivo Evaluation.

Contrast echocardiography (CE) ultrasound with microbubble contrast agents has significantly advanced our capability for assessment of cardiac function, including myocardium perfusion quantification. However, in standard CE techniques obtained with line by line scanning, the frame rate and image quality are limited. Recent research has shown significant frame-rate improvement in noncontrast cardiac imaging. In this work, we present and initially evaluate, both in vitro and in vivo, a high-frame-rate (HFR) CE imaging system using diverging waves and pulse inversion sequence. An imaging frame rate of 5500 frames/s before and 250 frames/s after compounding is achieved. A destruction-replenishment sequence has also been developed. The developed HFR CE is compared with standard CE in vitro on a phantom and then in vivo on a sheep heart. The image signal-to-noise ratio and contrast between the myocardium and the chamber are evaluated. The results show up to 13.4-dB improvement in contrast for HFR CE over standard CE when compared at the same display frame rate even when the average spatial acoustic pressure in HFR CE is 36% lower than the standard CE. It is also found that when coherent compounding is used, the HFR CE image intensity can be significantly modulated by the flow motion in the chamber.

Super-Resolution Axial Localization of Ultrasound Scatter Using Multi-Focal Imaging.

OBJECTIVE: This paper aims to develop a method for achieving micrometre axial scatterer localization for medical ultrasound, surpassing the inherent, pulse length dependence limiting ultrasound imaging. METHODS: The method, directly translated from cellular microscopy, is based on multi-focal imaging and the simple, aberration-dependent, image sharpness metric of a single point scatterer. The localization of a point scatterer relies on the generation of multiple overlapping sharpness curves, created by deploying three foci during receive processing, and by assessing the sharpness values after each acquisition as a function of depth. Each derived curve peaks around the receive focus and the unique position of the scatterer is identified by combining the data from all curves using a maximum likelihood algorithm with a calibration standard. RESULTS: Simulated and experimental ultrasound point scatter data show that the sharpness method can provide scatterer axial localization with an average accuracy down to 10.21 m ( 21) and with up to 11.4 times increased precision compared to conventional localization. The improvements depend on the rate of change of sharpness using each focus, and the signal to noise ratio in each image. CONCLUSION: Super-resolution axial imaging from optical microscopy has been successfully translated into ultrasound imaging by using raw ultrasound data and standard beamforming. SIGNIFICANCE: The normalized sharpness method has the potential to be used in scatterer localization applications and contribute in current super-resolution ultrasound imaging techniques.

Experimental performance assessment of the sub-band minimum variance beamformer for ultrasound imaging.

Recent progress in adaptive beamforming techniques for medical ultrasound has shown that current resolution limits can be surpassed. One method of obtaining improved lateral resolution is the Minimum Variance (MV) beamformer. The frequency domain implementation of this method effectively divides the broadband ultrasound signals into sub-bands (MVS) to conform with the narrow-band assumption of the original MV theory. This approach is investigated here using experimental Synthetic Aperture (SA) data from wire and cyst phantoms. A 7MHz linear array transducer is used with the SARUS experimental ultrasound scanner for the data acquisition. The lateral resolution and the contrast obtained, are evaluated and compared with those from the conventional Delay-and-Sum (DAS) beamformer and the MV temporal implementation (MVT). From the wire phantom the Full-Width-at-Half-Maximum (FWHM) measured at a depth of 52mm, is 16.7µm (0.08λ) for both MV methods, while the corresponding values for the DAS case are at least 24 times higher. The measured Peak-Side-lobe-Level (PSL) may reach -41dB using the MVS approach, while the values from the DAS and MVT beamforming are above -24dB and -33dB, respectively. From the cyst phantom, the power ratio (PR), the contrast-to-noise ratio (CNR), and the speckle signal-to-noise ratio (sSNR) measured at a depth of 30mm are at best similar for MVS and DAS, with values ranging between -29dB and -30dB, 1.94 and 2.05, and 2.16 and 2.27 respectively. In conclusion the MVS beamformer is not suitable for imaging continuous targets, and significant resolution gains were obtained only for isolated targets.

Influence of temperature, needle gauge and injection rate on the size distribution, concentration and acoustic responses of ultrasound contrast agents at high frequency.

This paper investigated the influence of needle gauge (19G and 27G), injection rate (0.85ml·min(-1), 3ml·min(-1)) and temperature (room temperature (RT) and body temperature (BT)) on the mean diameter, concentration, acoustic attenuation, contrast to tissue ratio (CTR) and normalised subharmonic intensity (NSI) of three ultrasound contrast agents (UCAs): Definity, SonoVue and MicroMarker (untargeted). A broadband substitution technique was used to acquire the acoustic properties over the frequency range 17-31MHz with a preclinical ultrasound scanner Vevo770 (Visualsonics, Canada). Significant differences (P<0.001-P<0.05) between typical in vitro setting (19G needle, 3ml·min(-1) at RT) and typical in vivo setting (27G needle, 0.85ml·min(-1) at BT) were found for SonoVue and MicroMarker. Moreover we found that the mean volume-based diameter and concentration of both SonoVue and Definity reduced significantly when changing from typical in vitro to in vivo experimental set-ups, while those for MicroMarker did not significantly change. From our limited measurements of Definity, we found no significant change in attenuation, CTR and NSI with needle gauge. For SonoVue, all the measured acoustic properties (attenuation, CTR and NSI) reduced significantly when changing from typical in vitro to in vivo experimental conditions, while for MicroMarker, only the NSI reduced, with attenuation and CTR increasing significantly. These differences suggest that changes in physical compression and temperature are likely to alter the shell structure of the UCAs resulting in measureable and significant changes in the physical and high frequency acoustical properties of the contrast agents under typical in vitro and preclinical in vivo experimental conditions.

Manufacturing of microcirculation phantoms using rapid prototyping technologies.

In this paper, we describe a method for the manufacturing of a microcirculation phantom that may be used to investigate hemodynamics using optics based methods. We made an Acrylonitrile Butadiene Styrene (ABS) negative mold, manufactured in a Fused Deposition Modelling (FDM) printer, embedded it in Polydimethysilioxane (PDMS) and dissolved it from within using acetone. We successfully made an enlarged three-dimensional (3D) network of microcirculation, and tested it using red blood cell (RBC) analogues. This phantom may be used for testing medical imaging technology.

High resolution depth-resolved imaging from multi-focal images for medical ultrasound.

An ultrasound imaging technique providing sub-diffraction limit axial resolution for point sources is proposed. It is based on simultaneously acquired multi-focal images of the same object, and on the image metric of sharpness. The sharpness is extracted by image data and presents higher values for in-focus images. The technique is derived from biological microscopy and is validated here with simulated ultrasound data. A linear array probe is used to scan a point scatterer phantom that moves in depth with a controlled step. From the beamformed responses of each scatterer position the image sharpness is assessed. Values from all positions plotted together form a curve that peaks at the receive focus, which is set during the beamforming. Selection of three different receive foci for each acquired dataset will result in the generation of three overlapping sharpness curves. A set of three calibration curves combined with the use of a maximum-likelihood algorithm is then able to estimate, with high precision, the depth location of any emitter fron each single image. Estimated values are compared with the ground truth demonstrating that an accuracy of 28.6 µm (0.13λ) is achieved for a 4 mm depth range.

The polydisperse acoustic signature of rigid microbubbles.

Microbubbles are used in medical ultrasound imaging as contrast agents to image the vascular bed under the mode of Ultrasound Contrast Imaging (UCI). The microbubble shell determines the acoustic response and hence the signal that is utilized to form the images in UCI. Single microbubble signals from BiSphere™ (POINT Biomedical, San Carlos, CA, USA) microbubbles were captured using a clinical ultrasound system. Three main typical responses of microbubbles were identified, a) full duration echo, b) echo with duration shorter than the incident pulse and c) echo that in part resembles that in (b) and in addition prior to that another short duration initial lower amplitude signal. These data corroborate that the shell structural and nanomechanical property provide the different responses at different microbubble sizes. These different signals present an opportunity for tracking the movement of well differentiated single microbubbles particularly with novel super-resolution imaging methods that require sparse microbubble populations.

The local effects of ovarian diathermy in an ovine model of polycystic ovary syndrome.

In order to develop a medical alternative to surgical ovarian diathermy (OD) in polycystic ovary syndrome (PCOS) more mechanistic information is required about OD. We therefore studied the cellular, molecular and vascular effects of diathermy on the ovary using an established ovine model of PCOS. Pregnant sheep were treated twice weekly with testosterone propionate (100 mg) from day 30-100 gestation. Their female offspring (n = 12) were studied during their second breeding season when the PCOS-like phenotype, with anovulation, is fully manifest. In one group (n = 4) one ovary underwent diathermy and it was collected and compared to the contralateral ovary after 24 hours. In another group a treatment PCOS cohort underwent diathermy (n = 4) and the ovaries were collected and compared to the control PCOS cohort (n = 4) after 5 weeks. Ovarian vascular indices were measured using contrast-enhanced ultrasound and colour Doppler before, immediately after, 24 hours and five weeks after diathermy. Antral follicles were assessed by immunohistochemistry and ovarian stromal gene expression by quantitative RT-PCR 24 hours and 5 weeks after diathermy. Diathermy increased follicular atresia (P<0.05) and reduced antral follicle numbers after 5 weeks (P<0.05). There was an increase in stromal CCL2 expression 24 hours after diathermy (P<0.01) but no alteration in inflammatory indices at 5 weeks. Immediately after diathermy there was increased microbubble transit time in the ovarian microvasculature (P = 0.05) but this was not seen at 24 hours. However 24 hours after diathermy there was a reduction in the stromal Doppler blood flow signal (P<0.05) and an increased ovarian resistance index (P<0.05) both of which persisted at 5 weeks (P<0.01; P<0.05). In the ovine model of PCOS, OD causes a sustained reduction in ovarian stromal blood flow with an increased ovarian artery resistance index associated with atresia of antral follicles.

In vitro acoustic characterization of three phospholipid ultrasound contrast agents from 12 to 43 MHz.

The acoustic properties of two clinical (Definity, Lantheus Medical Imaging, North Billerica, MA, USA; SonoVue, Bracco S.P.A., Milan, Italy) and one pre-clinical (MicroMarker, untargeted, Bracco, Geneva, Switzerland; VisualSonics, Toronto, ON, Canada) ultrasound contrast agent were characterized using a broadband substitution technique over the ultrasound frequency range 12-43 MHz at 20 ± 1°C. At the same number concentration, the acoustic attenuation and contrast-to-tissue ratio of the three native ultrasound contrast agents are comparable at frequencies below 30 MHz, though their size distributions and encapsulated gases and shells differ. At frequencies above 30 MHz, native MicroMarker has higher attenuation values and contrast-to-tissue ratios than native Definity and SonoVue. Decantation was found to be an effective method to alter the size distribution and concentration of native clinical microbubble populations, enabling further contrast enhancement for specific pre-clinical applications.

The ovine corpus luteum angiogenesis model: a tool for developing imaging technology.

Robust tools for the quantitation of perfusion are not fully developed using contrast enhanced ultrasound (CEUS). The ovine corpus luteum (CL) is a transient gland in the ovary that is formed to produce the hormone progesterone essential for maintenance of pregnancy. Importantly, it has a dense microvascular network with predictable and well-regulated angiogenic mechanisms. In a number of different experiments it was shown that this property may be used to investigate and refine imaging methodology. Using a Philips iU22 ultrasound scanner (Philips Medical Systems Corp, Seattle, WA) in contrast imaging mode it was shown that a highly controlled experiment may produce high levels of reproducibility in the transit of contrast with standard uncertainty below 10%. Also, compartmental kinetics models were tested. The use of prostaglandin F2alpha promotes an intense anti-angiogenesis, allowing monitoring with CEUS prior to and following the demise of the CL microvasculature within 24 hours. Finally, the robust angiogenic property of the CL during the oestrous cycle allows further refinement of CEUS in vivo. In conclusion, the CL offers an attractive changing vascular bed for assessing existing and developing new clinically relevant perfusion imaging methodology.

Automatic dissociation between microvasculature and larger vessels for ultrasound contrast imaging.

Microvasculature density (MVD) provides an established biomarker for the prognosis of numerous diseases associated with abnormal microvascular networks. The accurate, robust and timely assessment of MVD changes facilitates disease detection, treatment monitoring and patient stratification. Nevertheless, the current gold standard (PET) for MVD quantification is not used in clinical practice due to its high costs and potential health hazards. Contrast Enhanced Ultrasound (CEUS) imaging can provide an attractive alternative. However, the limited dissociation between larger vessels and microvasculature in the imaged tissues limits the accuracy and robustness of CEUS. This study proposed a novel, and fully automatic technique that dissociates larger vessels from microvasculature in CEUS imaged tissues. The ovine Corpus Luteum (CL) was used as an in vivo model for the development and assessment of the proposed technique.

A setup for the assessment of the effect of tubular confinement on the acoustic response of microbubbles.

Ultrasound contrast agents are gas filled microbubbles which produced enhanced echoes in ultrasound imaging thus allowing the acquisition of detailed information on the path of blood. It is theoretically known that the size of a vessel affects the behavior of a microbubble, which could potentially be used to discriminate different sized vessels. This information would be useful in the monitoring of neovascularization in tumor growth or treatment. However, currently it is not possible to identify the vessel diameter by any means of signal processing of microbubble echoes. In order to assess microbubble behavior when confined in tubes we compared the acoustic backscatter from biSphere™ microbubbles both free in water and flowing in 200 µm diameter tubes that are similar in size to arterioles. Experimental systems that allow the interrogation of individual microbubbles were designed and modified to allow investigation of both free microbubbles and those in tubes. Unprocessed single microbubble RF data were collected, allowing the calculation of both the fundamental and second harmonic components of the backscattered signal. Microbubbles confined in tubes had lower amplitude response compared to unconfined microbubbles. On consecutive insonations of the same microbubble, free microbubbles produced echoes above noise more often than confined microbubbles. This setup may be used to investigate microbubble behavior in a range of smaller tubes with diameters similar to capillaries thus enabling signal processing design for vessel differentiation.

Microbubble oscillations in capillary tubes.

In diagnostic medicine, microbubbles are used as contrast agents to image blood flow and perfusion in large and small vessels. The small vessels (the capillaries) have diameters from a few hundred micrometers down to less than 10 µ m. The effect of such microvessels surrounding the oscillating microbubbles is currently unknown, and is important for increased sensitivity in contrast diagnostics and manipulation of microbubbles for localized drug release. Here, oscillations of microbubbles in tubes with inner diameters of 25 µm and 160 ¿m are investigated using an ultra-high-speed camera at frame rates of ~12 million frames/s. A reduction of up to 50% in the amplitude of oscillation was observed for microbubbles in the smaller 25-µm tube, compared with those in a 160-µm tube. In the 25-µm tube, at 50 kPa, a 48% increase of microbubbles that did not oscillate above the noise level of the system was observed, indicating increased oscillation damping. No difference was observed between the resonance frequency curves calculated for microbubbles in 25-µm and 160-µm tubes. Although previous investigators have shown the effect of microvessels on microbubble oscillation at high ultrasound pressures, the present study provides the first optical images of low-amplitude microbubble oscillations in small tubes.

The speed of sound and attenuation of an IEC agar-based tissue-mimicking material for high frequency ultrasound applications.

This study characterized the acoustic properties of an International Electromechanical Commission (IEC) agar-based tissue mimicking material (TMM) at ultrasound frequencies in the range 10-47 MHz. A broadband reflection substitution technique was employed using two independent systems at 21°C ± 1°C. Using a commercially available preclinical ultrasound scanner and a scanning acoustic macroscope, the measured speeds of sound were 1547.4 ± 1.4 m∙s(-1) and 1548.0 ± 6.1 m∙s(-1), respectively, and were approximately constant over the frequency range. The measured attenuation (dB∙cm(-1)) was found to vary with frequency f (MHz) as 0.40f + 0.0076f(2). Using this polynomial equation and extrapolating to lower frequencies give values comparable to those published at lower frequencies and can estimate the attenuation of this TMM in the frequency range up to 47 MHz. This characterisation enhances understanding in the use of this TMM as a tissue equivalent material for high frequency ultrasound applications.

Nanomechanical properties of phospholipid microbubbles.

This study uses atomic force microscopy (AFM) force-deformation (F-Δ) curves to investigate for the first time the Young's modulus of a phospholipid microbubble (MB) ultrasound contrast agent. The stiffness of the MBs was calculated from the gradient of the F-Δ curves, and the Young's modulus of the MB shell was calculated by employing two different mechanical models based on the Reissner and elastic membrane theories. We found that the relatively soft phospholipid-based MBs behave inherently differently to stiffer, polymer-based MBs [Glynos, E.; Koutsos, V.; McDicken, W. N.; Moran, C. M.; Pye, S. D.; Ross, J. A.; Sboros, V. Langmuir2009, 25 (13), 7514-7522] and that elastic membrane theory is the most appropriate of the models tested for evaluating the Young's modulus of the phospholipid shell, agreeing with values available for living cell membranes, supported lipid bilayers, and synthetic phospholipid vesicles. Furthermore, we show that AFM F-Δ curves in combination with a suitable mechanical model can assess the shell properties of phospholipid MBs. The "effective" Young's modulus of the whole bubble was also calculated by analysis using Hertz theory. This analysis yielded values which are in agreement with results from studies which used Hertz theory to analyze similar systems such as cells.

Imaging of the ovine corpus luteum microcirculation with contrast ultrasound.

Ultrasound contrast agents have been the subject of microvascular imaging research. The sheep corpus luteum (CL) is a microvascular tissue that provides a natural angiogenic and antiangiogenic process, which changes during the luteal phase of the estrous cycle of the ewe. It can also be controlled and monitored endocrinologically, providing a very attractive in vivo model for the study and development of microvascular measurement. The perfusion of the fully developed CL between days 8 and 12 of the estrous cycle was studied in six ewes. A Philips iU22 ultrasound scanner (Bothell, WA, USA) with the linear array probe L9-3 was used to capture contrast-enhanced images after an intravenous bolus injection of 2.4 mL SonoVue (Bracco S.P.A., Milan, Italy). Time-intensity curves of a region of interest inside the CL were formed from linearized image data. A lagged-normal model to simulate the compartmental kinetics of the microvascular flow was used to fit the data, and the wash-in time was measured. Good contrast enhancement was observed in the CLs of all animals and the wash-in time averaged at 5.5 s with 9% uncertainty. The regression coefficient was highly significant for all fits. These data correlated with stained endothelial area in the histology performed postmortem. Two ewes were injected with prostaglandin F2alpha to induce CL regression, which resulted in an increase of wash-in time after a few hours. The CL of the ewe is thus proposed as an ideal model for the study and development of microvascular measurements using contrast ultrasound. Our initial results demonstrate a highly reproducible model for the study of the microvascular hemodynamics in a range of tissues and organs.