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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.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.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.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.ZIEL: Der Nachweis und die Differenzierung metastatischer Lymphknoten (LK) von den LK einer benignen Lymphadenopathie sind entscheidend für die Krebsdiagnose und -prognose, stellen jedoch eine klinische Herausforderung dar. Ein neuer Fortschritt der Superresolution-Ultraschall-Bildgebung (SRUS) durch Lokalisierung einzelner Mikrobläschen hat die Beugungsgrenze durchbrochen und eine nicht invasive In-vivo-Bildgebung der Gefäßmorphologie und Flussdynamik auf mikroskopischer Ebene ermöglicht. In dieser Studie stellen wir die Hypothese auf, dass SRUS quantitativen Markern ermöglicht, metastatische von benignen LK bei Patienten mit Lymphadenopathie zu unterscheiden.Klinische kontrastverstärkte Ultraschall-Bildsequenzen der LK von 6 Patienten mit Lymphknoten-Metastasen und von 4 mit gutartiger Lymphadenopathie wurden aufgenommen und bewegungskorrigiert. Diese wurden dann verwendet, um hochaufgelöste mikrovaskuläre Bilder und hochaufgelöste Geschwindigkeitskarten zu erstellen. Aus diesen SRUS-Bildern wurden morphologische und funktionelle Messwerte gewonnen, darunter die Dichte der Mikrogefäße, die fraktale Dimension, die mittlere Flussgeschwindigkeit und die Unregelmäßigkeit der lokalen Flussrichtung („Local Flow Direction Irregularity“, LFDI), die die Abweichung der lokalen Flussrichtung misst. Diese Messungen wurden zwischen pathologisch nachgewiesenen reaktiven und metastatischen LK verglichen.Unsere ersten Ergebnisse deuten darauf hin, dass der Unterschied beim Indikator Fluss-Unregelmäßigkeit (LFDI), der aus den SRUS-Bildern abgeleitet wird, zwischen den beiden Gruppen statistisch signifikant ist. Die LFDI ist bei metastatischen LK um 60% höher als bei reaktiven Knoten.Diese Pilotstudie zeigt die Durchführbarkeit der klinischen Superresolution-Ultraschall-Bildgebung bei Lymphknoten und das Potenzial der Nutzung der Unregelmäßigkeit lokaler Blutflussrichtungen, wie sie der SRUS für die Charakterisierung von LK bietet.

Visualizing vasculature beyond the diffraction limit can be achieved using ultrasound super-resolution imaging. Typically, ultrasound scanners model the target medium as homogeneous, assuming a constant speed-of-sound for time-of-flight based calculations. However, variations in ultrasound propagation velocity caused by varying tissue layers affect beamforming operations. This aberration is likely to have considerable effect on super-resolution ultrasound imaging at depth. Here we investigate the effect of aberration on super-resolution ultrasound localization error in soft tissues using simulations. Wave propagation using both linear, B-Mode, and nonlinear, pulse inversion, (PI) imaging acquisition through different tissue media was modelled from a linear array using k-Wave, and the resulting pulse-echo data from a microbubble located at 50 mm depth was beamformed. Media included a control homogeneous propagation medium, and aberrating media typical of liver imaging. Results indicated super-resolution ultrasound localisation errors increased with increasing aberration and were affected by both the imaging method and microbubble sizes. Average localisation errors for PI reached 749 µm axially and 986 µm laterally and 844 µm axially and 734 µm laterally for B-mode. The scale of these errors relative to micro-vascular structures of interest suggests that aberration will have considerable impact on super-resolution ultrasound performance and requires attention to ensure its success.

Coherent multi-transducer ultrasound (CoMTUS) creates an extended effective aperture through coherent combination of multiple arrays that results in significantly improved images, with enhanced resolution, extended field-of-view, and higher sensitivity. The approach has been previously validated in 2-D imaging using two linear arrays constrained into the same elevational plane. However, any misalignment between the arrays in the elevational plane could compromise the performance of the method. Extending CoMTUS to 3-D would generalise the framework and provide a mitigation for misalignment issues. In this study, CoMTUS is implemented and demonstrated for the first time in 3-D imaging using a pair of 2-D 256-element sparse arrays, which allows the channel count and the amount of data to be kept within feasible bounds. Simulated results show that, in comparison to a single array system using the same number of active elements, CoMTUS leads to significant improvements in resolution, contrast, and contrast-to-noise-ratio.

Cardiac catheterization is the gold standard for assessing cardiac pressures. However, this is an invasive and costly procedure. An alternative may be to use the subharmonic signal of microbubble ultrasound contrast agents to estimate pressure using echocardiography. The aim of this work was to investigate the subharmonic response of the ultrasound contrast agent SonoVue (Bracco Spa, Milan, Italy) to dynamic pressures in a flow phantom. SonoVue was circulated through a silicone tube using a peristaltic pump. The ULtrasound Advanced Open Platform (ULA-OP) was used with a cardiac phased array transducer to transmit a pulse-inversion sequence at 2.1 MHz. Radiofrequency data were recorded at five acoustic pressures (71–228 kPa peak-negative) to investigate the effects of pulse length (7-, 12- and 16-cycle pulses) and flow rate (150, 200 and 375 mL/min). The subharmonic (1.05 MHz) amplitude of SonoVue was calculated over a 40% bandwidth. A solid-state catheter recorded ground-truth pressures. Increasing pulse length improved the correlation coefficient between the subharmonic signal of SonoVue and pressure (r = 0.74 ± 0.14, 0.79 ± 0.06 vs. 0.86 ± 0.04 for 7-, 12- and 16-cycle pulses, respectively; $n$ = 3 at optimal acoustic pressure). The strong correlations with 16-cycle pulses persisted at different flow rates ( $r > 0.8$ ). This is the first report showing the ability of SonoVue to track pressure cycles five times faster than the average resting heart rate (376 cycles/min at 375 mL/min flow rate). SonoVue may potentially be used to assess diastolic and pulse pressures using ultrasound instead of cardiac catheterization.

Ultrasound super-resolution techniques use the response of microbubble contrast agents (MBs) 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 (CTR). MB responses were simulated based on the properties of Sonovue™ and using the Marmottant model. Non-linear propagation through tissue was modelled 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 a MB and tissue non-linear 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.

Coherent multi-transducer ultrasound (CoMTUS) imaging creates an extended effective aperture through coherent combination of multiple arrays. Such a large aperture, which dictates the image performance of the system, results in significantly improved images with enhanced resolution, extended field-of-view and higher sensitivity. This work investigates the role that the distribution of the individual arrays and the transmit sequence have in CoMTUS imaging performance. A parametric study is presented in which the geometry of the system, defined by two identical linear arrays, and the transmit sequence, using plane waves, are investigated. CoMTUS performance was assessed from the point-spread-function, in terms of first and second side lobe amplitudes and lateral resolution. Results suggest that CoMTUS performance can be enhanced by optimizing the position of the multiple arrays and the transmit sequence.

Medical image analysis is becoming increasingly accessible in the clinic. Computed tomography (CT) or magnetic resonance imaging (MRI) scans are usually post-processed to generate a 3-D visualization of the human body for surgical assistance or extract quantitative data to provide additional diagnostic information. Three dimensional super-resolution ultrasound (SR US) imaging can provide similar information at a micro-level without the high cost or ionising radiation. In this study, we implemented a high volumetric-rate 3-D SR US imaging with a 2-D spiral-shaped array and imaged an in vitro microvascular structure. From the 3-D SR US images clinically relevant parameters, such as microvascular flow rate, microvessel density and tortuosity, were extracted and compared with the ground truth.

Current localization-based super-resolution ultrasound imaging requires a low concentration of flowing microbubbles to visualize microvasculature beyond the diffraction limit and acquisition is slow. Recent studies have shown that Acoustic Wave Sparsely Activated Localization Microscopy (AWSALM) using phase change nanodroplets can generate fast or even real-time super-resolution imaging. However, the optimal experimental conditions to activate the droplets for generating the super-resolution images are still unknown, especially the activation ultrasound amplitude or mechanical index (MI). The aim of this study is to investigate the effect of the activation MI on the repeated activation of decafluorobutane nanodroplets in vivo . Noninvasive ultrasound data acquisition on a rabbit kidney using the AWSALM pulse sequence was repeated for different activation MIs. It was found that the droplet activation was not observed in the rabbit kidney at an MI of 1.1. The activation of droplet started at an activation MI of 1.3. The contrast of activated droplet signals is maximized at an MI of 1.5 and decreased when the activation MI was increased above 1.5. Such understanding of the effects of activation MI could help improve droplet-base fast super-resolution imaging.

The fabella is a sesamoid bone usually located in the tendon of the lateral head of the gastrocnemius muscle, behind the knee joint. Prevalence rates in human populations vary widely with an average of 42.5% people having a fabella. Clinically, it is associated with a number of knee ailments, most notably the osteoarthritis of the knee and generalized knee pain (i.e ., fabella syndrome). As the function of the fabella remains unknown, the biomechanical consequences of fabella presence/absence can only be speculated. Successfully detecting the fabella, measuring its size and determining its shape, are off importance for clinical and evolutionary researchers. In this work, we compare plane wave imaging with conventional focused imaging and evaluate their performance for detecting and characterizing the fabella.

Despite medical ultrasound being ubiquitous in clinical practice, its usefulness is limited by low resolution images with a restricted field of view and penetration depth. Those key limitations, which are especially prevalent when imaging deep in the body and are more pronounced in an increasingly obese population, reduce sensitivity of diagnostic ultrasound. Fundamentally, these limitations are constrained by the extent of the transmitting and receiving apertures. Recently, we have developed coherent multi-transducer ultrasound (CoMTUS), which allows the use of multiple ultrasound arrays as one large effective aperture, to overcome known limitations and improve the ultrasound imaging performance. In this study, CoMTUS feasibility for in vivo imaging is tested, for the first time, in a foetal phantom and in vivo in a healthy adult volunteer. Preliminary results show that, in comparison to a single-probe image and to the fused image obtained combining the two images independently acquired by both arrays, CoMTUS leads to improved images with an extended field of view and more detailed structures.

Visualisation of the vasculature in clinical ultrasound has been greatly enhanced with the development of microbubbles as ultrasound contrast agents. These contrast agents have gained increasing use due to their safe profile, improved stability in the bloodstream and ability to provide a unique response even at low transmit powers. The availability of new generations of engineered ultrasound contrast agents, along with advances in specialised contrast-specific imaging methods to exploit their unique response, has facilitated the progress of contrast-enhanced ultrasound imaging (CEUS) into a routine diagnostic procedure. A comprehensive understanding of the physics of microbubbles and their interaction with ultrasound will continue to improve current imaging techniques and the development of new ones.

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.

The structure of microvasculature cannot be resolved using conventional ultrasound imaging due to the fundamental diffraction limit at clinical ultrasound frequencies. It is possible to overcome this resolution limitation by localizing individual microbubbles through multiple frames and forming a super-resolved 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 imaging is prone to motion artefacts as other imaging modalities based on multiple acquisitions are. This study investigates the feasibility of a two-stage motion estimation method, which is a combination of affine and non-rigid estimation, for super-resolution ultrasound imaging. Firstly, the motion correction accuracy of the proposed method is evaluated using simulations with increasing complexity of motion. A mean absolute error of 12.2 μm was achieved in simulations for the worst case scenario. The motion correction algorithm was then applied to a clinical dataset to demonstrate its potential to enable in vivo super-resolution ultrasound imaging in the presence of patient motion. The size of the identified microvessels from the clinical super-resolution images were measured to assess the feasibility of the two-stage motion correction method, which reduced the width of the motion blurred microvessels approximately 1.5-fold.

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 (r(2) = 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.

The measurement of cardiac and aortic pressures enable 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 radiofrequency 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.

A coherent multi-transducer ultrasound imaging system (CoMTUS) enables an extended effective aperture through coherent combination of multiple transducers. The resulting larger effective aperture improves the ultrasound imaging performance. The optimal beamforming parameters, which include the transducer locations and the average speed of sound in the medium, are deduced by maximizing the coherence of the received radio frequency data by cross-correlation. In this technique, the detection of multiple isolated point-like targets in the overlap of the insonated regions is mandatory to determine the relative probe-to-probe position. This study proposes the use of microbubbles to generate the point-like targets that the CoMTUS approach requires. The first phantom images produced using CoMTUS and microbubbles are presented here.

In this study, a photoacoustic super-resolution imaging technique was developed through imaging the activation of Cyanine 7.5-coated phase-change nanodroplets using a preclinical photoacoustic imaging system and localizing the activated droplets. As a proof-of-concept experiment, photoacoustic images of flowing dye-coated nanodroplets in microfluidic channels were obtained with a cylindrically focused curved-array. Experimental results showed that super-resolution images can resolve the microfluidic channels which cannot be resolved by conventional beamformed images. The results also showed that the dye-coated phase-change nanodroplets can be optically activated in vivo and the activation signals can be separated from the image background by applying singular value decomposition filtering, and be used for further super-localization processing. Such nanodroplets can offer better biocompatibility, as well as more flexible and controllable droplet activation rates, with potential for super-resolution imaging of static and extravascular targets, compared to existing contrast agents used in existing localization-based photoacoustic super-resolution imaging techniques.

Nanodroplets are on-demand ultrasound contrast agents. They can remain in circulation longer in vivo than mi-crobubbles and can be spatiotemporally and selectively activated to provide contrast when required. Perfluorocarbon nanodroplets have been used for conventional 2D ultrasound imaging and different types of nanodroplets have been studied and characterized by researchers regarding their suitability for imaging. However, the use of nanodroplets for 3D imaging and their activation using a 2D ultrasound probe has not been reported. In this study, we investigate the nanodroplet activation using a 2D ultrasound imaging probe within clinical safety limits.

Current localization-based super-resolution ultrasound imaging requires a low concentration of flowing microbubbles to visualize microvasculature beyond the diffraction limit and acquisition is slow. Nanodroplets offer a promising solution as they can be sparsely activated and deactivated on-demand. In this study, acoustic wave sparsely-activated localization microscopy (AWSALM) using activation and deactivation of nanodroplets, an acoustic counterpart of photo-activated localization microscopy (PALM) which is less dependent on agent concentration and the presence of flow, is demonstrated for super-resolution imaging in deep tissues in vivo. An in vivo super-resolution image of a rabbit kidney is obtained in 1.1 seconds using AWSALM, where micro-vessels with apparent sizes far below the half-wavelength of 220 µm were visualized. This preliminary result demonstrates the feasibility of applying AWSALM for in vivo super-resolution imaging.

It has been previously established that the subharmonic signal of microbubble ultrasound contrast agents follows an inverse linear relationship with the ambient hydrostatic pressure. However, contradictory results have been reported for SonoVue (Bracco Spa, Milan, Italy). Therefore, the aim of this study was to investigate the subharmonic response of SonoVue across a range of physiologically-relevant hydrostatic pressures (10–220 mmHg), and to compare it with Sonazoid (GE, Oslo, Norway). A modified GE Logiq 9 scanner was used to acquire the subharmonic signals from Sonazoid and SonoVue in a sealed water tank. In addition, the Ultrasound Advanced Open Platform (ULA-OP) was used to acquire the subharmonic signal of SonoVue at 0–200 mmHg in a cell culture cassette over approximately 3 min. Sonazoid showed an inverse linear relationship between subharmonic amplitude and hydrostatic pressure (slope −0.11 dB/mmHg; r = −0.81), as expected. SonoVue exhibited an increase in subharmonic amplitude from 0–100 mmHg hydrostatic pressure (slope 0.06 dB/mmHg, r = 0.81), a plateau between 100–140 mmHg, and a decrease from 140–220 mmHg (slope −0.26 dB/mmHg, r = −0.98). The subharmonic amplitude of SonoVue increased over 3 min at 0 and 25 mmHg hydrostatic pressure, did not change with time at 50–100 mmHg, and decreased over time at 125–200 mmHg. However, the effects of time on the subharmonic amplitude of SonoVue were smaller than the effects of hydrostatic pressure. The subharmonic response of SonoVue to hydrostatic pressure differs from Sonazoid and other microbubble ultrasound contrast agents.

One of the crucial challenges in the application of ultrasound super-resolution imaging using microbubble contrast agents to the clinic is the long acquisition time. Recently acoustic wave sparsely activated localization microscopy (AWSALM) was developed to activate, image, and destroy nanodroplets to achieve ultrasound super-resolution imaging. The activation and destruction of nanodroplets using focused pulses in AWSALM can generate new localization signals without relying on flow and shows the potential to achieve faster ultrasound super-resolution. However, the technique requires optimization to achieve good activation efficiency with the minimum amount of activation time.This work investigates how the different activation strategies affect the activation time and efficiency. A range of activation sequences with different F-numbers and spacing between transmit foci are used and their activation efficiency and the data acquisition time are quantified. The results show that sweeping the focus of the activation pulse with an F-number of 0.4 and a step size of 2.5 wavelengths can generate the highest droplet activation. By using a larger or smaller spacing between consecutive focused activation beams generated lower contrast, which suggests that densely spaced activation pulses may destroy the activated nanodroplets generated by the previous activation pulse. In summary, this study demonstrates that the rate of activation of nanodroplets can be increased by optimizing the F-number and the sweep step size. This finding can inform the development of droplet-based super-resolution imaging to further minimize the data acquisition time.

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 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 9-angle plane waves acquired in 3 milliseconds at a pulse repetition frequency of 3000 Hz. To allow microbubbles sufficient time to move between consequent compounded volumetric frames, a 7-millisecond delay was introduced after each volume acquisition. This reduced the effective volume acquisition speed to 100 Hz and the total acquired data size by 3.3-fold. Localization-based 3-D super-resolution images of two touching sub-wavelength tubes were generated from 6000 volumes acquired in 60 seconds. In conclusion, this work demonstrates the feasibility of 3D super-resolution imaging and super-resolved velocity mapping using a customized 2D sparse array transducer.

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 $\lambda $ /10 to $\lambda $ /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 (

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

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.

Radical orchifunicolectomy has traditionally been the main clinical treatment for small testicular masses (STMs); however STMs represent a constantly increasing and often incidental finding. Since many of them result benign, a more conservative testis-sparing surgery was proposed, but it requires a preliminary differentiation between benign and malignant masses: this however remains challenging. Although common understanding in radiology and oncology is that perfusion patterns might provide a useful information about the type of masses, no guidelines or consensus is available for the differentiation of STMs. We propose to build a dictionary of relevant perfusion patterns, extracted using non-negative matrix factorization on pixel-wise time-intensity curves from contrast-enhanced ultrasound data. When data from a lesion are reconstructed using this dictionary, a vector containing the frequency of utilization of each pattern can be used as a tissue signature. Using this signature, a support vector machine classifier has been trained, and the cross validated accuracy reached 100% in our pilot cohort.

Localization-based super-resolution imaging requires accurate detection of spatially isolated microbubbles. The reason for this requirement is that interfering or overlapping signals resulting from multiple microbubbles within the resolution limit can cause position errors. In addition to this, noise and artefacts (e.g. residual tissue signal after tissue-microbubble separation) further reduce the quality and hence the spatial resolution in SR imaging. Therefore, correctly identifying the echoes as noise, single microbubble, multiple microbubbles, or artefact is important.In this study, the use of fast classification methods for identification and rejection of non-single microbubble echoes were demonstrated. Most commonly used supervised classification methods, including Decision Trees, Discriminant Analysis, Logistic Regression, Support Vector Machine, Ensembles, k-Nearest Neighbors, and Naive Bayes, were implemented for filtering artefacts and noise in super-resolution ultrasound images. Results showed that the Ensemble method, explicitly designed to deal with unbalanced data, achieved the best result since most of the localized events are true microbubbles, which is typical for super-resolution imaging datasets.

Localization-based ultrasound super-resolution imaging can visualize the microvascular structure beyond the diffraction limit but is sensitive to contrast concentration and requires acquisition for seconds. Low-boiling-point phase-change nanodroplets achieve super-resolved images with subsecond temporal resolution through activation and destruction with high frame rate plane waves in real-time.

Visualizating vasculature beyond the diffraction limit can be achieved using ultrasound super-resolution (USR). Typically, ultrasound (US) scanners model the target medium as homogeneous, assuming a constant speed-of-sound for time-of-flight based calculations. However, variations in US propagation velocity caused by varying tissue layers affect beamforming operations. This aberration is likely to have considerable effect on USR accuracy when imaging at depth. Here we investigate the effect of aberration on USR localization accuracy. Wave propagation through different tissue media was modelled from a linear transducer using k-Wave, and the resulting pulse-echo data from a point scatterer located at 45mm depth was beamformed. Media included a control homogeneous propagation medium, and aberrating media typical of liver imaging. Aberration effects were estimated using RMS arrival-time fluctuations (ATF) and energy-level fluctuations (ELF) at the scatterer location. Signals were extracted and localized with existing localization techniques. Results indicated USR localisation accuracy decreased with increasing aberration. Axial localisation errors reached 836.6 mm for an ATF 30.9 ns. Furthermore, errors increased with decreasing frequency from 4 to 3 MHz. The scale of these errors relative to micro-vascular structures of interest suggests that aberration will have considerable impact on USR performance and requires attention to ensure its success.

A crucial challenge in the application of ultrasound super-resolution imaging using microbubbles to the clinic is the long acquisition time together with the motion during the data acquisition. In this study, fast acoustic wave sparsely activated localization microscopy (fast-A WSALM)is developed to simultaneously image, activate and deactivate octafluoropropane nanodroplets during high-frame-rate planewave ultrasound imaging to achieve ultrasound super-resolution images on a sub-second timescale. This work demonstrates in vitro fast-A Wsalmof a non-flow 200-micron-tube phantom. Firstly, experimental results show that the octafluoropropane nanodroplets can be activated by the plane-wave pulses, whereas acoustic droplet vaporization has only been achieved using focused single element transducers or linear-array probes with focus-wave transmission in previous literature. The activation of nanodroplets via plane-waves without the need of using focused-transmit waves enables a faster imaging and activation acquisition. Second, the results show the fast-AWSALM can give a better estimation of the tube diameter (190 um)where standard B-mode ultrasound image cannot (550 um), In summary, this study demonstrates the potential of fast-A WSALM, a super-resolution techniques using nanodroplets, which can generate super-resolution images in milliseconds and does not require flow or a precise control of contrast agent concentration.

Assessment of complex and disordered tumour vasculature requires full 3D visualization. Ultrasound super-resolution techniques are able to image microvascular structure and flow beyond the diffraction limit. Existing demonstrations have been predominantly 2D, where the elevational resolution remains restricted to around the millimeter range, while 3D demonstrations have either used mechanical scanning, or have required customized or state-of-the-art research systems to achieve true super-resolution in the third dimension. In this study, 3D super-resolution and velocity tracking is demonstrated in vitro using an ultrasound imaging system currently available in the clinic. This was performed at 1.25 MHz transmit frequency, with a frame rate of 54 Hz in contrast enhanced imaging mode. Three-dimensional super-resolved volumetric imaging of a twisted micro-vessel phantom was demonstrated at 3.5 cm depth, where between 66-70% of localizations where estimated to fall within the vessel internal diameter. Demonstration of 3D ultrasound super-resolution using a system currently available in the clinic demonstrates a fast route for clinical translation and application. In the future, 3D localization using microbubble signal onset could allow considerably improved microvascular visualization to aid early disease detection, diagnosis, and intervention for micro-vascular related diseases like cancer.

Acoustic super-resolution (SR) has the potential to visualize microvasculature by localizing individual microbubble (MB) signals. Currently, all detected signals are processed and localized identically. However, the MB point spread function (PSF) is not independent of its surroundings. Despite accuracy on the order of microns being required, it is currently not possible to quantify error that may be introduced due to variation in the MB responses. This work combines high frame rate plane wave ultrasound acquisition with a coincident optical microscope visualizing the SR imaging of a 200 µm cellulose tube. An adjustable aperture has been introduced into the optical microscope to extend the optical depth of field over the phantom. The results showed that the introduction of the aperture enabled modest extension of the depth of field over 50 µm about the optical focus. Modelling and experimental verification found that, at a flow rate of 15 µl/min, MBs could only be detected over the top 70 µm of the tube phantom - further reducing the required depth of field. The simultaneous optical and acoustic data suggested that many fewer MBs acoustically contribute to the SR image than can be observed in the optical FOV. Investigations incorporating a ground truth, like this one, will allow sources of error to be identified, quantified and limited.

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.

Photo-activated localization microscopy (PALM) has revolutionized the field of fluorescence microscopy by breaking the diffraction limit in spatial resolution. In this study, “acoustic wave sparsely activated localization microscopy (AWSALM),” an acoustic counterpart of PALM, is developed to super-resolve structures which cannot be resolved by conventional B-mode imaging. AWSALM utilizes acoustic waves to sparsely and stochastically activate decafluorobutane nanodroplets by acoustic vaporization and to simultaneously deactivate the existing vaporized nanodroplets via acoustic destruction. In this method, activation, imaging, and deactivation are all performed using acoustic waves. Experimental results show that sub-wavelength micro-structures not resolvable by standard B-mode ultrasound images can be separated by AWSALM. This technique is flow independent and does not require a low concentration of contrast agents, as is required by current ultrasound super resolution techniques. Acoustic activation and deactivation can be controlled by adjusting the acoustic pressure, which remains well within the FDA approved safety range. In conclusion, this study shows the promise of a flow and contrast agent concentration independent super-resolution ultrasound technique which has potential to be faster and go beyond vascular imaging.

© 2018 IEEE. Super-resolution ultrasound imaging has been so far achieved in 3-D by mechanically scanning a volume with a linear probe, by co-aligning multiple linear probes, by using multiplexed 3- D clinical ultrasound systems, or by using 3-D ultrasound research systems. In this study, a 2-D sparse array was designed with 512 elements according to a density-tapered 2-D spiral layout and optimized to reduce the sidelobes of the transmitted beam profile. High frame rate volumetric imaging with compounded plane waves was performed using two synchronized ULA-OP256 systems. Localization-based 3-D super-resolution images of two touching sub-wavelength tubes were generated from a 120 second acquisition. Print on Demand (PoD) ISBN: 978-1-5386-3426-4 ispartof: IEEE International Ultrasonics Symposium, IUS vol:2018-October ispartof: 2018 IEEE International Ultrasonics Symposium (IUS) location:Kobe, Japan date:22 Oct - 25 Oct 2018 status: Published online

Ultrasound super-resolution imaging can be achieved by localizing spatially isolated microbubble contrast agents over multiple imaging frames. In vivo images with resolutions of ∼10-20 microns in deep tissue have been demonstrated. The technique has the potential to revolutionize the way micro-circulation can be visualized and quantified, and has implications in a wide range of clinical applications including cancer, diabetes and beyond. In this paper we describe the principle of the technique with in vivo results demonstrating the superior resolution achieved compared with existing ultrasound imaging. We also discuss the challenges and opportunities in the area of 3D imaging including, imaging speed, tissue motion and microbubble localization errors.

Sub-diffraction imaging, also known as ultrasound localization microscopy, is a novel method that can overcome the fundamental diffraction limit by localizing spatially isolated microbubbles. This method requires the use of a low concentration of microbubbles to ensure that they are spatially isolated. For in vivo microvascular imaging, especially for cancer tissue with high microvascular density, spatial isolation cannot be always achieved, since vessels are close to each other and the speed of flow is slow. This study proposes a frequency decomposition method that uses the polydisperse nature of commercial contrast agents to separate spatially non-isolated microbubbles with different acoustic signatures. Zero-phase filters were applied to ensure that there is no relative phase delay between decomposed signals. Results showed that a super-resolution image after frequency decomposition can be generated with three times lower number of acquisitions without sacrificing image quality.

Physiologically important pressures in the heart and aorta are currently assessed with invasive pressure catheters. The subharmonic signal from microbubble ultrasound contrast agents, however, may be exploited to estimate pressures non-invasively. The objective of this work was (i) to develop a static phantom from commercially-available components for easy replication across different laboratories, and (ii) to investigate the subharmonic response of the ultrasound contrast agent SonoVue (Bracco Spa, Milan, Italy) at physiological pressures within this phantom. A phantom capable of maintaining 0-200 mmHg static pressures was developed using a cell culture cassette with Luer connections. SonoVue was added (0.4 μL/mL) and radiofrequency data were recorded on the ULtrasound Advanced Open Platform (ULA-OP) from 3.5-100% scanner acoustic output levels (transmit frequency 5 MHz, 16-cycle pulse, pulse-inversion). Signal processing was performed to extract the mean subharmonic amplitude in a 1 MHz bandwidth (2-3 MHz). A single growth phase between 75-325 kPa peak-to-peak acoustic pressures was observed at ambient pressure (0 mmHg). Within this growth phase, SonoVue exhibited an increase in subharmonic amplitude from 0-75 mmHg hydrostatic pressure, a plateau between 75-125 mmHg, and a decrease from 125-200 mmHg. The maximum sensitivity of SonoVue to hydrostatic pressure up to 75 mmHg was observed at 152 kPa peak-to-peak acoustic pressure (0.13 dB/mmHg, r2=0.99). This increase in subharmonic signal could have arisen from compression-only behavior as predicted by the Marmottant model, and may have clinical applications in estimating diastolic pressures non-invasively.

Clinical implementation of super-resolution (SR) ultrasound imaging requires accurate single microbubble detection, and would benefit greatly from automation in order to minimize time requirements and user dependence. We present a machine learning based post-processing tool for the application of SR ultrasound imaging, where we utilize superpixelation and support vector machines (SVMs) for foreground detection and signal differentiation.

Recently, acoustic super-resolution (SR) imaging has allowed visualization of microvascular structure and flow beyond the diffraction limit through the localization of many isolated microbubble signals. Each bubble position is typically estimated by calculating the centroid, finding a local maximum, or finding the peak of a 2-D Gaussian function fit. However, the backscattered signal from a microbubble depends not only on diffraction characteristics of the waveform, but also on the bubble behavior in the acoustic field, which if not accounted for, may cause localization errors.

The structure of microvasculature cannot be resolved using clinical B-mode or contrast-enhanced ultrasound (CEUS) imaging due to the fundamental diffraction limit at clinical ultrasound frequencies. It is possible to overcome this resolution limitation by localizing individual microbubbles through multiple frames and forming a super-resolved image. However, ultrasound super-resolution creates its unique problems since the structures to be imaged are on the order of 10s of μm. Tissue movement much larger than 10 μm is common in clinical imaging, which can significantly reduce the accuracy of super-resolution images created from microbubble locations gathered through hundreds of frames. This study investigated an existing motion estimation algorithm from magnetic resonance imaging for ultrasound super-resolution imaging. Its correction accuracy is evaluated using simulations with increasing complexity of motion. Feasibility of the method for ultrasound super-resolution in vivo is demonstrated on clinical ultrasound images. For a chosen microvessel, the super-resolution image without motion correction achieved a sub-wavelength resolution; however after the application of proposed two-stage motion correction method the size of the vessel was reduced to half.

Changes in microvascular structure and flow is of clinical importance in the study of a number of disease processes such as cancer and diabetes. Ultrasound is often the primary imaging procedure performed to determine appropriate treatment or surgery for testicular lesions. Currently, however, differentiation and diagnosis of both benign and malignant testicular tumours such as seminomas, leydig cell tumours and lymphomas are often challenging. Contrast-enhanced ultrasound (CEUS) has been used to aid their characterisation [1]. There are, however, a variety of benign testicular lesions that can mimic testicular malignancies. Ultrasound super-resolution (US-SR) techniques have been able to visualise vascular structures in vitro and in vivo beyond the diffraction limit by localizing individual microbubble signals. In this work, we aim to apply US-SR processing to clinical data to aid diagnostic confidence.

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.

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 $900~\mu \text{m}$ 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.

Many diseases, such as those associated with diabetes, coronary heart disease, and the growth and metastasis of tumors exhibit architectural changes in microvascular structure, as well as variations in vascular flow. Current clinical imaging modalities such as MRI, PET, CT and Ultrasound cannot easily resolve the microvasculature. Although optical light microscopy is able to resolve structures on the scale of microvasculature, the restricted imaging depth of this approach is a major limitation. Thus, there is a clinical need for the development of new imaging techniques that can fill this resolution gap.