Super-Resolution Ultrasound Localization Microscopy of Microvascular Structure and Flow for Distinguishing Metastatic Lymph Nodes - An Initial Human Study.

PURPOSE: Detecting and distinguishing metastatic lymph nodes (LNs) from those with benign lymphadenopathy are crucial for cancer diagnosis and prognosis but remain a clinical challenge. A recent advance in super-resolution ultrasound (SRUS) through localizing individual microbubbles has broken the diffraction limit and tracking enabled in vivo noninvasive imaging of vascular morphology and flow dynamics at a microscopic level. In this study we hypothesize that SRUS enables quantitative markers to distinguish metastatic LNs from benign ones in patients with lymphadenopathy. MATERIALS AND METHODS: Clinical contrast-enhanced ultrasound image sequences of LNs from 6 patients with lymph node metastasis and 4 with benign lymphadenopathy were acquired and motion-corrected. These were then used to generate super-resolution microvascular images and super-resolved velocity maps. From these SRUS images, morphological and functional measures were obtained including micro-vessel density, fractal dimension, mean flow speed, and Local Flow Direction Irregularity (LFDI) measuring the variance in local flow direction. These measures were compared between pathologically proven reactive and metastasis LNs. RESULTS: Our initial results indicate that the difference in the indicator of flow irregularity (LFDI) derived from the SRUS images is statistically significant between the two groups. The LFDI is 60% higher in metastatic LNs compared with reactive nodes. CONCLUSION: This pilot study demonstrates the feasibility of super-resolution ultrasound for clinical imaging of lymph nodes and the potential of using the irregularity of local blood flow directions afforded by SRUS for the characterization of LNs.

Super-resolution Ultrasound Imaging.

The majority of exchanges of oxygen and nutrients are performed around vessels smaller than 100 µm, allowing cells to thrive everywhere in the body. Pathologies such as cancer, diabetes and arteriosclerosis can profoundly alter the microvasculature. Unfortunately, medical imaging modalities only provide indirect observation at this scale. Inspired by optical microscopy, ultrasound localization microscopy has bypassed the classic compromise between penetration and resolution in ultrasonic imaging. By localization of individual injected microbubbles and tracking of their displacement with a subwavelength resolution, vascular and velocity maps can be produced at the scale of the micrometer. Super-resolution ultrasound has also been performed through signal fluctuations with the same type of contrast agents, or through switching on and off nano-sized phase-change contrast agents. These techniques are now being applied pre-clinically and clinically for imaging of the microvasculature of the brain, kidney, skin, tumors and lymph nodes.

3-D Super-Resolution Ultrasound Imaging With a 2-D Sparse Array.

High-frame-rate 3-D ultrasound imaging technology combined with super-resolution processing method can visualize 3-D microvascular structures by overcoming the diffraction-limited resolution in every spatial direction. However, 3-D super-resolution ultrasound imaging using a full 2-D array requires a system with a large number of independent channels, the design of which might be impractical due to the high cost, complexity, and volume of data produced. In this study, a 2-D sparse array was designed and fabricated with 512 elements chosen from a density-tapered 2-D spiral layout. High-frame-rate volumetric imaging was performed using two synchronized ULA-OP 256 research scanners. Volumetric images were constructed by coherently compounding nine-angle plane waves acquired at a pulse repetition frequency of 4500 Hz. Localization-based 3-D super-resolution images of two touching subwavelength tubes were generated from 6000 volumes acquired in 12 s. Finally, this work demonstrates the feasibility of 3-D super-resolution imaging and super-resolved velocity mapping using a customized 2-D sparse array transducer.

Optimal Control of SonoVue Microbubbles to Estimate Hydrostatic Pressure.

The measurement of cardiac and aortic pressures enables diagnostic insight into cardiac contractility and stiffness. However, these pressures are currently assessed invasively using pressure catheters. It may be possible to estimate these pressures less invasively by applying microbubble ultrasound contrast agents as pressure sensors. The aim of this study was to investigate the subharmonic response of the microbubble ultrasound contrast agent SonoVue (Bracco Spa, Milan, Italy) at physiological pressures using a static pressure phantom. A commercially available cell culture cassette with Luer connections was used as a static pressure chamber. SonoVue was added to the phantom, and radio frequency data were recorded on the ULtrasound Advanced Open Platform (ULA-OP). The mean subharmonic amplitude over a 40% bandwidth was extracted at 0-200-mmHg hydrostatic pressures, across 1.7-7.0-MHz transmit frequencies and 3.5%-100% maximum scanner acoustic output. The Rayleigh-Plesset equation for single-bubble oscillations and additional hysteresis experiments were used to provide insight into the mechanisms underlying the subharmonic pressure response of SonoVue. The subharmonic amplitude of SonoVue increased with hydrostatic pressure up to 50 mmHg across all transmit frequencies and decreased thereafter. A decreasing microbubble surface tension may drive the initial increase in the subharmonic amplitude of SonoVue with hydrostatic pressure, while shell buckling and microbubble destruction may contribute to the subsequent decrease above 125-mmHg pressure. In conclusion, a practical operating regime that may be applied to estimate cardiac and aortic blood pressures from the subharmonic signal of SonoVue has been identified.

Poisson Statistical Model of Ultrasound Super-Resolution Imaging Acquisition Time.

A number of acoustic super-resolution techniques have recently been developed to visualize microvascular structure and flow beyond the diffraction limit. A crucial aspect of all ultrasound (US) super-resolution (SR) methods using single microbubble localization is time-efficient detection of individual bubble signals. Due to the need for bubbles to circulate through the vasculature during acquisition, slow flows associated with the microcirculation limit the minimum acquisition time needed to obtain adequate spatial information. Here, a model is developed to investigate the combined effects of imaging parameters, bubble signal density, and vascular flow on SR image acquisition time. We find that the estimated minimum time needed for SR increases for slower blood velocities and greater resolution improvement. To improve SR from a resolution of λ /10 to λ /20 while imaging the microvasculature structure modeled here, the estimated minimum acquisition time increases by a factor of 14. The maximum useful imaging frame rate to provide new spatial information in each image is set by the bubble velocity at low blood flows (<150 mm/s for a depth of 5 cm) and by the acoustic wave velocity at higher bubble velocities. Furthermore, the image acquisition procedure, transmit frequency, localization precision, and desired super-resolved image contrast together determine the optimal acquisition time achievable for fixed flow velocity. Exploring the effects of both system parameters and details of the target vasculature can allow a better choice of acquisition settings and provide improved understanding of the completeness of SR information.

3D Super-Resolution US Imaging of Rabbit Lymph Node Vasculature in Vivo by Using Microbubbles.

Background Variations in lymph node (LN) microcirculation can be indicative of metastasis. The identification and quantification of metastatic LNs remains essential for prognosis and treatment planning, but a reliable noninvasive imaging technique is lacking. Three-dimensional super-resolution (SR) US has shown potential to noninvasively visualize microvascular networks in vivo. Purpose To study the feasibility of three-dimensional SR US imaging of rabbit LN microvascular structure and blood flow by using microbubbles. Materials and Methods In vivo studies were carried out to image popliteal LNs of two healthy male New Zealand white rabbits aged 6-8 weeks. Three-dimensional, high-frame-rate, contrast material-enhanced US was achieved by mechanically scanning with a linear imaging probe. Individual microbubbles were identified, localized, and tracked to form three-dimensional SR images and super-resolved velocity maps. Acoustic subaperture processing was used to improve image contrast and to generate enhanced power Doppler and color Doppler images. Vessel size and blood flow velocity distributions were evaluated and assessed by using Student paired t test. Results SR images revealed microvessels in the rabbit LN, with branches clearly resolved when separated by 30 µm, which is less than half of the acoustic wavelength and not resolvable by using power or color Doppler. The apparent size distribution of most vessels in the SR images was below 80 µm and agrees with micro-CT data, whereas most of those detected with Doppler techniques were larger than 80 µm in the images. The blood flow velocity distribution indicated that most of the blood flow in rabbit popliteal LN was at velocities lower than 5 mm/sec. Conclusion Three-dimensional super-resolution US imaging using microbubbles allows noninvasive nonionizing visualization and quantification of lymph node microvascular structures and blood flow dynamics with resolution below the wave diffraction limit. This technology has potential for studying the physiologic functions of the lymph system and for clinical detection of lymph node metastasis. Published under a CC BY 4.0 license. Online supplemental material is available for this article.

Fast Acoustic Wave Sparsely Activated Localization Microscopy (fast-AWSALM): Ultrasound Super-Resolution using Plane-Wave Activation of Nanodroplets.

Localization-based ultrasound super-resolution imaging using microbubble contrast agents and phase-change nano-droplets has been developed to visualize microvascular structures beyond the diffraction limit. However, the long data acquisition time makes the clinical translation more challenging. In this study, fast acoustic wave sparsely activated localization microscopy (fast-AWSALM) was developed to achieve super-resolved frames with sub-second temporal resolution, by using low-boiling-point octafluoropropane nanodroplets and high frame rate plane waves for activation, destruction, as well as imaging. Fast-AWSALM was demonstrated on an in vitro microvascular phantom to super-resolve structures that could not be resolved by conventional B-mode imaging. The effects of the temperature and mechanical index on fast-AWSALM was investigated. Experimental results show that sub-wavelength micro-structures as small as 190 lm were resolvable in 200 ms with plane-wave transmission at a center frequency of 3.5 MHz and a pulse repetition frequency of 5000 Hz. This is about a 3.5 fold reduction in point spread function full-width-half-maximum compared to that measured in conventional B-mode, and two orders of magnitude faster than the recently reported AWSALM under a non-flow/very slow flow situations and other localization based methods. Just as in AWSALM, fast-AWSALM does not require flow, as is required by current microbubble based ultrasound super resolution techniques. In conclusion, this study shows the promise of fast-AWSALM, a super-resolution ultrasound technique using nanodroplets, which can generate super-resolution images in milli-seconds and does not require flow.

Investigation of Microbubble Detection Methods for Super-Resolution Imaging of Microvasculature.

Ultrasound super-resolution techniques use the response of microbubble (MB) contrast agents to visualize the microvasculature. Techniques that localize isolated bubble signals first require detection algorithms to separate the MB and tissue responses. This work explores the three main MB detection techniques for super-resolution of microvasculature. Pulse inversion (PI), differential imaging (DI), and singular value decomposition (SVD) filtering were compared in terms of the localization accuracy, precision, and contrast-to-tissue ratio. MB responses were simulated based on the properties of Sonovue and using the Marmottant model. Nonlinear propagation through tissue was modeled using the k-Wave software package. For the parameters studied, the results show that PI is most appropriate for low frequency applications, but also most dependent on transducer bandwidth. SVD is preferable for high frequency acquisition where localization precision on the order of a few microns is possible. PI is largely independent of flow direction and speed compared to SVD and DI, so is appropriate for visualizing the slowest flows and tortuous vasculature. SVD is unsuitable for stationary MBs and can introduce a localization error on the order of hundreds of microns over the speed range 0-2 mm/s and flow directions from lateral (parallel to probe) to axial (perpendicular to probe). DI is only suitable for flow rates >0.5 mm/s or as flow becomes more axial. Overall, this study develops an MB and tissue nonlinear simulation platform to improve understanding of how different MB detection techniques can impact the super-resolution process and explores some of the factors influencing the suitability of each.

Two-Stage Motion Correction for Super-Resolution Ultrasound Imaging in Human Lower Limb.

The structure of microvasculature cannot be resolved using conventional ultrasound (US) imaging due to the fundamental diffraction limit at clinical US frequencies. It is possible to overcome this resolution limitation by localizing individual microbubbles through multiple frames and forming a superresolved image, which usually requires seconds to minutes of acquisition. Over this time interval, motion is inevitable and tissue movement is typically a combination of large- and small-scale tissue translation and deformation. Therefore, super-resolution (SR) imaging is prone to motion artifacts as other imaging modalities based on multiple acquisitions are. This paper investigates the feasibility of a two-stage motion estimation method, which is a combination of affine and nonrigid estimation, for SR US imaging. First, the motion correction accuracy of the proposed method is evaluated using simulations with increasing complexity of motion. A mean absolute error of 12.2 was achieved in simulations for the worst-case scenario. The motion correction algorithm was then applied to a clinical data set to demonstrate its potential to enable in vivo SR US imaging in the presence of patient motion. The size of the identified microvessels from the clinical SR images was measured to assess the feasibility of the two-stage motion correction method, which reduced the width of the motion-blurred microvessels to approximately 1.5-fold.

3-D Motion Correction for Volumetric Super-Resolution Ultrasound Imaging.

Motion during image acquisition can cause image degradation in all medical imaging modalities. This is particularly relevant in 2-D ultrasound imaging, since out-of-plane motion can only be compensated for movements smaller than elevational beamwidth of the transducer. Localization based super-resolution imaging creates even a more challenging motion correction task due to the requirement of a high number of acquisitions to form a single super-resolved frame. In this study, an extension of two-stage motion correction method is proposed for 3-D motion correction. Motion estimation was performed on high volumetric rate ultrasound acquisitions with a handheld probe. The capability of the proposed method was demonstrated with a 3-D microvascular flow simulation to compensate for handheld probe motion. Results showed that two-stage motion correction method reduced the average localization error from 136 to 18 µm.

Microbubble Axial Localization Errors in Ultrasound Super-Resolution Imaging.

Acoustic super-resolution imaging has allowed the visualization of microvascular structure and flow beyond the diffraction limit using standard clinical ultrasound systems through the localization of many spatially isolated microbubble signals. The determination of each microbubble position is typically performed by calculating the centroid, finding a local maximum, or finding the peak of a 2-D Gaussian function fit to the signal. However, the backscattered signal from a microbubble depends not only on diffraction characteristics of the waveform, but also on the microbubble behavior in the acoustic field. Here, we propose a new axial localization method by identifying the onset of the backscattered signal. We compare the accuracy of localization methods using in vitro experiments performed at 7-cm depth and 2.3-MHz center frequency. We corroborate these findings with simulation results based on the Marmottant model. We show experimentally and in simulations that detecting the onset of the returning signal provides considerably increased accuracy for super-resolution. Resulting experimental cross-sectional profiles in super-resolution images demonstrate at least 5.8 times improvement in contrast ratio and more than 1.8 times reduction in spatial spread (provided by 90% of the localizations) for the onset method over centroiding, peak detection, and 2-D Gaussian fitting methods. Simulations estimate that these latter methods could create errors in relative bubble positions as high as at these experimental settings, while the onset method reduced the interquartile range of these errors by a factor of over 2.2. Detecting the signal onset is, therefore, expected to considerably improve the accuracy of super-resolution.

3-D In Vitro Acoustic Super-Resolution and Super-Resolved Velocity Mapping Using Microbubbles.

Standard clinical ultrasound (US) imaging frequencies are unable to resolve microvascular structures due to the fundamental diffraction limit of US waves. Recent demonstrations of 2-D super-resolution both in vitro and in vivo have demonstrated that fine vascular structures can be visualized using acoustic single bubble localization. Visualization of more complex and disordered 3-D vasculature, such as that of a tumor, requires an acquisition strategy which can additionally localize bubbles in the elevational plane with high precision in order to generate super-resolution in all three dimensions. Furthermore, a particular challenge lies in the need to provide this level of visualization with minimal acquisition time. In this paper, we develop a fast, coherent US imaging tool for microbubble localization in 3-D using a pair of US transducers positioned at 90°. This allowed detection of point scatterer signals in 3-D with average precisions equal to [Formula: see text] in axial and elevational planes, and [Formula: see text] in the lateral plane, compared to the diffraction limited point spread function full-widths at half-maximum of 488, 1188, and [Formula: see text] of the original imaging system with a single transducer. Visualization and velocity mapping of 3-D in vitro structures was demonstrated far beyond the diffraction limit. The capability to measure the complete flow pattern of blood vessels associated with disease at depth would ultimately enable analysis of in vivo microvascular morphology, blood flow dynamics, and occlusions resulting from disease states.

A Temporal and Spatial Analysis Approach to Automated Segmentation of Microbubble Signals in Contrast-Enhanced Ultrasound Images: Application to Quantification of Active Vascular Density in Human Lower Limbs.

Contrast-enhanced ultrasound (CEUS) using microbubble contrast agents has shown great promise in visualising and quantifying active vascular density. Most existing approaches for vascular density quantification using CEUS are calculated based on image intensity and are susceptible to confounding factors and imaging artefact. Poor reproducibility is a key challenge to clinical translation. In this study, a new automated temporal and spatial signal analysis approach is developed for reproducible microbubble segmentation and quantification of contrast enhancement in human lower limbs. The approach is evaluated in vitro on phantoms and in vivo in lower limbs of healthy volunteers before and after physical exercise. In this approach, vascular density is quantified based on the relative areas microbubbles occupy instead of their image intensity. Temporal features of the CEUS image sequences are used to identify pixels that contain microbubble signals. A microbubble track density (MTD) measure, the ratio of the segmented microbubble area to the whole tissue area, is calculated as a surrogate for active capillary density. In vitro results reveal a good correlation (r2 = 0.89) between the calculated MTD measure and the known bubble concentration. For in vivo results, a significant increase (129% in average) in the MTD measure is found in lower limbs of healthy volunteers after exercise, with excellent repeatability over a series of days (intra-class correlation coefficient = 0.96). This compares to the existing state-of-the-art approach of destruction and replenishment analysis on the same patients (intra-class correlation coefficient ≤0.78). The proposed new approach shows great potential as an accurate and highly reproducible clinical tool for quantification of active vascular density.

In vivo acoustic super-resolution and super-resolved velocity mapping using microbubbles.

The structure of microvasculature cannot be resolved using standard clinical ultrasound (US) imaging frequencies due to the fundamental diffraction limit of US waves. In this work, we use a standard clinical US system to perform in vivo sub-diffraction imaging on a CD1, female mouse aged eight weeks by localizing isolated US signals from microbubbles flowing within the ear microvasculature, and compare our results to optical microscopy. Furthermore, we develop a new technique to map blood velocity at super-resolution by tracking individual bubbles through the vasculature. Resolution is improved from a measured lateral and axial resolution of 112 µm and 94 µ m respectively in original US data, to super-resolved images of microvasculature where vessel features as fine as 19 µm are clearly visualized. Velocity maps clearly distinguish opposing flow direction and separated speed distributions in adjacent vessels, thereby enabling further differentiation between vessels otherwise not spatially separated in the image. This technique overcomes the diffraction limit to provide a noninvasive means of imaging the microvasculature at super-resolution, to depths of many centimeters. In the future, this method could noninvasively image pathological or therapeutic changes in the microvasculature at centimeter depths in vivo.