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Individuals with diabetes at risk of developing diabetic kidney disease (DKD) are challenging to identify using currently available clinical methods. Prognostic accuracy and initiation of treatment could be improved by a quantification of the renal microvascular rarefaction and the increased vascular tortuosity during the development of DKD. Super-resolution ultrasound (SRUS) imaging is an in vivo technique capable of visualizing blood vessels at sizes below 75 µm. This preclinical study aimed to investigate the alterations in renal blood vessels’ density and tortuosity in a type 2 diabetes rat model, Zucker diabetic fatty (ZDF) rats, as a prediction of DKD. Lean age-matched Zucker rats were used as controls. A total of 36 rats were studied, subdivided into ages of 12, 22, and 40 weeks. Measured albuminuria indicated the early stage of DKD, and the SRUS was compared with the ex vivo micro-computed tomography (µCT) of the same kidneys. Assessed using the SRUS imaging, a significantly decreased cortical vascular density was detected in the ZDF rats from 22 weeks of age compared to the healthy controls, concomitant with a significantly increased albuminuria. Already by week 12, a trend towards a decreased cortical vascular density was found prior to the increased albuminuria. The quantified vascular density in µCT corresponded with the in vivo SRUS imaging, presenting a consistently lower vascular density in the ZDF rats. Regarding vessel tortuosity, an overall trend towards an increased tortuosity was present in the ZDF rats. SRUS shows promise for becoming an additional tool for monitoring and prognosing DKD. In the future, large-scale animal studies and human trials are needed for confirmation.

A row-column beamformation algorithm is presented, which yields the output from the conventional delay-and-sum algorithm while reducing the number of operations by order of magnitude. The proposed method uses that in a row-column synthetic aperture sequence, the low-resolution volumes (LRV) have approximately constant image values along the elevation axis. It is, thus, possible to reconstruct the entire LRV from a single cross-section by modeling the positions with constant image values. As such, the proposed method consists of two stages. The first stage beamforms a low-resolution image per emission using the conventional approach. The second stage reconstructs the LRVs with one interpolation per voxel. Lastly, a high-resolution volume (HRV) is obtained by summing the LRVs across all emissions. The proposed algorithm was evaluated on measured data acquired using a 6 MHz 128+128 Vermon row-column probe and a Verasonics Vantage system. The proposed method beamformed a $100 \times 100 \times 200$ HRV consisting of 48 LRVs at a volume rate of 38 Hz. This was 9.23 times faster than a published GPU implementation of the conventional approach and real-time volumetric beamformation was achieved with a pulse repetition frequency of up to 1805 Hz. The output from the conventional and proposed beamformer was visually indistinguishable, and their point spread function's width heccurate interpolation requiright at -6 dB and -20 dB deviated less than 0.5%. This demonstrates that the number of operations of the conventional row-column beamformer can be significantly reduced with a negligible impact on image quality.

Obesity is a risk factor of chronic kidney disease (CKD), leading to alterations in the renal vascular structure. This study tested if renal vascular density and tortuosity was quantifiable in vivo in obese rats using microbubble-based super-resolution ultrasound imaging. The kidneys of two 11-week-old and two 20-week-old male obese Zucker rats were compared with age-matched male lean Zucker rats. The super-resolution ultrasound images were manually divided into inner me-dulla, outer medulla, and cortex, and each area was subdivided into arteries and veins. We quanti-fied vascular density and tortuosity, number of detected microbubbles, and generated tracks. For comparison, we assessed glomerular filtration rate, albumin/creatinine ratio, and renal histology to evaluate CKD. The number of detected microbubbles and generated tracks varied between animals and significantly affected quantification of vessel density. In areas with a comparable number of tracks, density increased in the obese animals, concomitant with a decrease in glomerular filtration rate and an increase in albumin/creatinine ratio, but without any pathology in the histological stain-ing. The results indicate that super-resolution ultrasound imaging can be used to quantify structural alterations in the renal vasculature. Techniques to generate more comparable number of microbub-ble tracks and confirmation of the findings in larger-scale studies are needed.

Super-resolution ultrasound imaging (SRUS) is a branch of ultrasound techniques aiming to image and quantify the vasculature beyond the diffraction limit [1]. Going beyond the diffraction limit of conventional ultrasound entails the possibility of imaging the microvasculature, namely arterioles, venules, and maybe even the smallest vessels in the body: the capillaries. In one of the main SRUS techniques, also called ultrasound localization microscopy, isolated microbubbles from ultrasound contrast agents are used to acquire data for SRUS image formation. Super-resolution ultrasound imaging using isolated microbubbles was inspired by one of the Nobel prize-winning approaches for super-resolution microscopy [2]. In one of these approaches, the ability to turn the fluorescence of single molecules on and off was used. By capturing numerous images of the same object, each image with a different group of molecules fluorescently turned on and superposing the resultant image stack, a super-resolved microscopy image, i. e., an image showing structures below the diffraction limit of light, could be created. Likewise, the SRUS images are created by superposing thousands of successive ultrasound images of isolated microbubbles as they move through the vasculature. More specifically, the SRUS images are created using a series of post-processing steps. After scanning the organ or tissue of interest, the sparsely distributed intravascular microbubbles must be detected. Detection can be done with, e. g., contrast-enhancing sequences, such as pulse inversion or amplitude modulation, or with singular value decomposition (SVD) techniques [3]. Next, the single microbubbles are isolated and localized [4]. The precision of this localization is a critical step in obtaining super-resolution [5]. Instead of merely superposing each of the microbubble localizations, as done in super-resolution microscopy, the movements of the microbubbles as they follow the bloodstream between frames are used

One of the integral parts of super-resolution ultrasound imaging (SRI) is particle tracking. This paper presents tracking for a new approach for SUper Resolution ultrasound imaging using Erythrocytes (SURE), which uses the erythrocytes as the target instead of fragile microbubbles. The acquisition of the SURE data can be accomplished in seconds due to the abundance of erythrocytes as targets. The nearest-neighbor (NN) algorithm was used to track erythrocytes. The erythrocyte targets were tracked to create SURE intensity map by three NN trackers with a constraint on the maximum velocities of 20, 40, 80 mm/s. By combining the outputs of three trackers, and inserting them into one map, and also using an image fusion method based on discrete wavelet transform for fusion the intensity maps, it was demonstrated that the combination of trajectories from different velocities and fusion of intensity maps carried more information from all the maps.

Super resolution (SR) imaging is currently conducted using fragile ultrasound contrast agents. This precludes using the full acoustic pressure range, and the distribution of bubbles has to be sparse for them to be isolated for SR imaging. Images have to be acquired over minutes to accumulate enough positions for visualizing the vas- culature. A new method for SUper Resolution imaging using the Erythrocytes (SURE) as targets is introduced, which makes it possible to maximize the emitted pressure for good signal-to-noise ratios. The abundant number of erythrocyte targets make acquisition fast, and the SURE images can be acquired in seconds. A Verasonics Vantage 256 scanner was used in combination with a GE L8-18iD linear array probe operated at 10 MHz for a wavelength of 150 m. A 12 emissions synthetic aperture ultrasound sequence was employed to scan the kidney of a Sprague-Dawley rat for 24 seconds to visualize its vasculature. An ex vivo micro-CT image using the contrast agent Microfil was also acquired at a voxel size of 22.6 m for validating the SURE images. The SURE image revealed vessels with a size down to 29 fim, five times smaller than the ultrasound wavelength, and the dense grid of vessels in the full kidney was reliably shown for scan times between 1 to 24 seconds. Visually the SURE images revealed the same vasculature as the micro-CT images. SURE images are acquired in seconds rather than minutes without contrast injection for easy clinical use, and they can be measured at full regulatory levels for pressure, intensity, and probe temperature.

One of the integral parts of super-resolution ultrasound imaging (SRI) is particle tracking, and researchers are trying to improve SRI with different tracking methods. This paper presents tracking for a new approach for SUper Resolution ultrasound imaging using Erythrocytes (SURE), which uses the erythrocytes as the target instead of fragile microbubbles (MBs). The acquisition of the SURE data can be accomplished in seconds due to the abundance of erythrocytes as targets. The nearest-neighbor (NN) algorithm was used to track erythrocytes, since this is one of the successful approaches for MB tracking in SRI. The erythrocyte targets were tracked to create SURE intensity map by three NN trackers with a constraint on the maximum velocities of 20, 40, 80 mm/s. Each tracker generated a different track map that depict different details. By combining the outputs of three trackers, and inserting them into one map, it was demonstrated that the combination of trajectories from different velocities carried more information from all the maps. Also, an image fusion method using discrete wavelet transform is applied on the intensity maps of these three dfferent velocities. Qualitative and quantitative experiments were conducted to exhibit the enhancements of the combined and the fused intensity map. Comparing three intensity maps with their combination and fusion and also a magnified area showed the fused one had better visual appearance than the others. Standard deviation (SD), average gradient (AG), entropy (H) and spatial frequency (SF) were used for quantitative comparison. According to SD, AG, H, and SF, the fused intensity map had 10%, 46%, 2%, and 46% higher scores compared to the combined intensity map. Also, the fused intensity map had 19% and 22% higher scores compared to the intensity map with maximum velocities of 80 mm/s according to AG and SF, respectively.

Current super resolution imaging is conducted using ultrasound contrast agents, where a sparse distribution ofbubbles must be employed to separate individual targets. Thesparse targets make the acquisition time long in the range of1 to 10 minutes, and therefore demands an accurate motioncorrection over a long time. The employment of a contrastagent also lowers MI to below 0.2 to not disrupt the bubbles,with a corresponding lower signal-to-noise ratio in the images.A new method, SURE (SUper Resolution ultrasound imagingusing Erythrocytes), where erythrocytes are used as targets, issuggested to alleviate these problems. Perfused tissues containan abundance of targets, and the full clinical pressure rangecan be used. It is hypothesized that super resolution imagingbelow the diffraction limit can be attained in seconds usingSURE imaging. A SURE processing pipeline was developed withmodules for beamforming, tissue motion estimation, alignment,singular value decomposition for echo canceling, and subsequentpeak detection in the speckle pattern. The detected peaks weresummed in a high-resolution image for yielding the SURE image.Data were acquired using a 10 MHz linear array GE L10-18i probe (150 µm wavelength) and a Verasonics Vantage 256scanner. A synthetic aperture scan sequence with 12 emissionswas employed at a pulse repetition frequency of 5 kHz for a 417Hz frame rate. Kidneys of Sprague-Dawley rats were scanned for24 seconds and RF data stored for off-line processing. The excisedkidneys were micro-CT scanned for 11 hours for generatingreference maps of the vasculature with a voxel size of 21 µm.SURE images revealed vessels with sizes down to 50 µm. Fourierring correlations between independent images measured for 12s revealed a resolution between 25 to 49 µm, demonstrating thesuper resolution capability of the method. The SURE images areobtained in 1 to 12 seconds, demand no injection of intravenouscontrast agents, and can use the full pressure and intensityrange allo

Microbubble (MB) tracking is an integral part of super-resolution ultrasound imaging by providing sharper images and enabling velocity estimation. Tracking the MBs from the last to the first frame can generate different trajectories than tracking from the first to the last frame, when the next positions of a track depends on its previous positions, e.g., in Kalman-based methods. Our hypothesis is that tracking in a forward-backward manner can increase the overall tracking performance. In simulations, MB positions with a parabolic flow profile were generated inside two tubes. Three different tracking methods, including nearest-neighbor, Kalman, and hierarchical Kalman, were investigated. Using the proposed forward-backward strategy, all estimated velocity profiles for all trackers were improved and were closer to the actual velocity profiles with an improvement between 28% to 40% in the relative standard deviation (RSD) of the velocity values over 10 cross-sections of the tubes. A Sprague Dawley rat kidney was scanned for 10 minutes using a BK5000 scanner and X18L5s transducer, which is a linear array probe with 150 elements. The tracking results from the in vivo experiments showed that the combined image of the forward and backward tracks had 35% additional unique track positions. It showed a clear visual enhancement in the super-resolved velocity map. Overall, the improvement in visual aspects and velocity estimates suggest forward-backward strategy as an upgrade for Kalman-based trackers.

Velocity is one of the clinically interesting parameters. A number of studies have shown the ability of super-resolution (SR) ultrasound imaging to visualize this parameter using velocity maps. However, manual separation of the velocity estimates for arteries from veins can be quite demanding. This study used the anatomical knowledge of rat kidneys for automatic classification of arterial and venous blood velocities in SR images and measured their variations in the medullary regions of four healthy Sprague-Dawley rat kidneys. The measurements were conducted using a modified bk5000 scanner (BK Medical, Herlev, Denmark) and a BK 9009 linear array probe with a pulse amplitude modulation scheme. Ten minutes of acquired B-mode and contrast images with frame-rate of 54 Hz were processed using a SR processing pipeline. The micro-bubble trajectories were filtered using coarse anatomy labels for classification of arterial and venous flow. The velocity estimates of separated arterioles and venules of the outer medulla showed separation of data in all rats. A Wilcoxon test showed that this difference was statistically significant (p=0.002). Considering the sample size for this study, the t-distributions predicted that the median velocity in the OM arterioles and venules were in the range of 0.84 ± 0.09 mm/s and 0.70 ± 0.07 mm/s with 95% confidence. The result showed how the blood flow in outer medulla arterioles and venules of rat kidneys can be automatically distinguished using the known anatomical information about the renal vasculature.

In vivo monitoring of the microvasculature is relevant since diseases such as diabetes, ischemia, or cancer cause microvascular impairment. Super-resolution ultrasound imaging allows in vivo examination of the microvasculature by detecting and tracking sparsely distributed intravascular microbubbles over a minute-long period. The ability to create detailed images of the renal vasculature of Sprague-Dawley rats using a modified clinical ultrasound platform was investigated in this study. Additionally, we hypothesized that early ischemic damage to the renal microcirculation could be visualized. After a baseline scan of the exposed kidney, 10 rats underwent clamping of the renal vein (n = 5) or artery (n = 5) for 45 min. The kidneys were rescanned at the onset of clamp release and after 60 min of reperfusion. Using a processing pipeline for tissue motion compensation and microbubble tracking, super-resolution images with a very high level of detail were constructed. Image filtration allowed further characterization of the vasculature by isolating specific vessels such as the ascending vasa recta with a 15-20 μm diameter. Using the super-resolution images alone, it was only possible for six assessors to consistently distinguish the healthy renal microvasculature from the microvasculature at the onset of vein clamp release. Future studies will aim at attaining quantitative estimations of alterations in the renal microvascular blood flow using super-resolution ultrasound imaging.

Tracking plays an important role in super-resolution (SR) ultrasound imaging, as it improves the quality and sharpness of the final SR images. Moreover, tracking enables quantification of clinically important parameters, such as blood flow velocity. However, the tracking performance degrades in the presence of complex particle patterns and localization uncertainty due to noise and motion. This work presents and discusses multiple approaches for tracking evaluation and compares a nearestneighbor (NN) with a Kalman tracker through simulations and an in vivo experiment. It is shown that in the presence of a localization uncertainty with a standard deviation (SD) of λ/5, the bias and SD of the velocity estimates reach -1.04 ± 0.9 mm/s and -0.12 ± 0.72 mm/s in the NN and Kalman tracker, respectively (relative to the peak velocity of 10 mm/s). The precision of individual track positions is estimated for an in vivo experiment as 37.95 ± 21.37 µm and 23.9 ± 11.82 µm for the NN and Kalman trackers, respectively. The results indicate that the Kalman tracker achieves a better velocity estimation and reduces localization uncertainty.

Super-resolution ultrasound imaging (SRI) can visualize and quantify changes in the microvasculature. Metabolic syndrome is associated with hyperlipidemia that affect different organs, including the kidneys. Ex vivo studies have shown glomerular injury in Obese Zucker rats (OZR) over time. If in vivo SRI can diagnose renal disease before it becomes evident with current measures, earlier treatment can be initiated to postpone the onset of renal complications in persons with metabolic syndrome. The overall aim of this study is to investigate whether SRI can detect early microvascular changes in the kidneys of rats with metabolic syndrome. The rats presented in this work were scanned at an early age to get a baseline scan prior to further studies. In this work an 11-week-old OZR and a healthy agematched Zucker rat were investigated. During open surgery, the left kidney was scanned for 10 min using a modified BK5000 scanner (BK Medical, Denmark), a fixed X18L5s transducer and intravenously administered SonoVue (1:10). Images were obtained using interleaved contrast (amplitude modulation) and B-mode sequences with focused beam transmission (6 MHz, 50 Hz, MI: 0.2). An in-house tool was used to track microbubble (MB) movements between frames, in order to estimate the MB velocities, which were measured in a large region of the cortex and the outer medulla. This is the first time SRI has been used on the kidneys of rats with Metabolic syndrome. Both the cortex and the medulla were well-perfused with MBs, and no morphological differences in the microvasculature were found between the two rats. The thickness of the cortex and the medulla was almost identical; cortex 1.8 mm, medulla 8 mm, craniocaudal length 2.0 vs. 1.9 cm (healthy vs. OZR). The same was true regarding the MB velocities (median (IQR) in mm/s) for healthy vs. OZR; cortex 0.75 (3.51) vs. 0.65 (2.64) and medulla 0.75 (0.32) vs. 0.62 (0.30). The results show that SRI can visualize the renal microvasculature of a yo

In vivo visualization of the microvasculature is feasible with super-resolution ultrasound imaging (SRI), but the method needs more affirmative data before clinical use. The kidneys have a rich vasculature, and microvascular dysfunction decreases the kidney function. Therefore, detection of subtle renal microvascular changes could benefit patients with renal disease. We hypothesized that our SRI setup can visualize the microvascular network of two healthy rat kidneys and subsequently demonstrate microvascular flow changes immediately after ischemia- reperfusion. The left kidney of two male Sprague-Dawley rats was scanned during laparotomy using a customized BK5000 scanner, an X18L5s transducer, and interleaved contrast and B-mode sequences with focused beam transmission. Images were acquired over 10 min using SonoVue (1:10) as the contrast agent. After a baseline scan, one rat had the renal vein clamped, while the other rat had the renal artery clamped, both for 45 min. The kidneys were rescanned immediately after clamp release and after 60 min of reperfusion. Motion correction was applied before microbubble (MB) detection. The characteristic renal microvascular structure was visualized with anatomical distinction between the dense cortical vascular network and the straight vessels of the medulla. Immediately after vein clamp release, almost no MBs perfused the medullary vessels. After 60 min of reperfusion, MBs refilled the renal vascular bed, but with a slower velocity compared with the baseline scan. The results after artery clamping were subtler, with more MBs perfusing the medulla immediately after clamp release compared with the vein clamping. The results imply that this SRI setup can evaluate different stages of ischemic kidney disease in rats as it can visualize the entire renal vascular bed and differentiate the pattern of reperfusion in two types of ischemic injury.

OBJECTIVES: Adult studies have proven ultrasound elastography as a validated measure of liver fibrosis. The present study aimed to review the available literature on ultrasound elastography in children to evaluate the ability of the method to distinguish healthy from fibrotic liver tissue and investigate whether cutoff values for liver fibrosis in children have been established.METHODS: A literature search was performed in MEDLINE, EMBASE, the Cochrane Library, and Web of Science to identify studies on ultrasound elastography of the liver in children. Only original research articles in English concerning ultrasound elastography in children with and without liver disease, younger than 18 years, were included. All reference lists of the included articles were hand-searched for further references.RESULTS: Twenty-seven articles were included. Elastography in children without liver disease was investigated in 14 studies and were comparable to those existing for adults. Twelve studies compared elastography with liver biopsy in children with liver disease and found that cirrhosis was correctly diagnosed, whereas it was more difficult to assess severe fibrosis correctly. For the distinction between no, mild, and moderate fibrosis in children with liver disease the method was less accurate. Ultrasound elastography was able to differentiate between children with and without liver fibrosis. In children without liver disease ultrasound, elastography showed consistent liver stiffness values comparable to those found in adults. No fibrosis-specific cutoffs were proposed.CONCLUSIONS: Ultrasound elastography was able to diagnose cirrhosis, distinguish healthy from fibrotic liver tissue, and showed consistent liver stiffness values in children without liver disease.