Microvascular Imaging with Super-Resolution Ultrasound

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 to create trajectories that can reveal microbubble velocity and direction [6] [7] [8] [9] [10]. Lastly, another essential difference between super-resolved microscopy and ultrasound is motion. In order to localize the microbubbles precisely, it is necessary to compensate for the motion that stems from, e. g., breathing and heart beating during scanning [11] [12].

The resulting trajectory-based images, created from localizing and tracking the isolated microbubbles, are used to reveal physiological or pathological changes in the vasculature. The majority of studies that have been published are preclinical studies, many of which have investigated technical feasibilities and developments. As microvascular disease can occur anywhere in the body, SRUS has been applied to various anatomical structures, from the eyes to the heart to the prostate [13] [14] [15]. However, the largest areas of interest have been the vasculature of the brain, the kidneys, and malignant tumors. In the brain, SRUS has been used to evaluate age-related vascular alterations, which revealed that microbubble velocities were slower and vessels more tortuous in old mouse brains compared with younger ones [16]. It has also been used to measure cerebral arterial pulsatility in mice to improve our understanding of the effects of increased pulse pressure on the brain’s microvasculature [17]. As a last example, SRUS has been acquired through an intact human skull at the temporal acoustic window [18], demonstrating cerebrovascular hemodynamics, including turbulent flow in an aneurism and chaotic flow in collateral arteries in a person with Moya Moya-like disease. As for the kidneys, a number of studies have demonstrated renal vascular changes related to disease. For example, unlike sham-operated mice, mice with unilateral ischemia-reperfusion-induced chronic kidney disease had decreased vascular density and increased tortuosity in the renal cortex [19]. In another study, a vasodilator-induced decrease in microbubble velocity was found in the cortex and the deeper-lying medulla of rat kidneys [20], and lastly, increased microbubble velocity was found in the cortical radial arteries of hypertensive rats [21]. The cortical vessels of a human kidney have also been visualized with SRUS [22]. In that study, the various human organs and lesions were imaged, including the liver in a healthy state and during liver failure, a pancreatic tumor, and a breast tumor. As for cancer, a higher number of studies have investigated the vasculature of tumors, including using different vascular parameters to distinguish malignant tumors with different vascular phenotypes from each other [23] or to distinguish tumor vessels from healthy ones [24]. Additionally, the effect of treatment on the tumor vessels has been investigated as an early marker for treatment response [25]. Besides primary tumor vasculature, a recently published study investigated whether lymph nodes with metastasis could be distinguished from reactive ones using SRUS in humans [26]. The study showed that metastatic lymph nodes had a more irregular blood flow, measured as the variance in flow direction in a given area, compared with the reactive lymph nodes.

The studies mentioned above comprise only a small selection of the many studies that have been published with SRUS since the first journal papers with in vivo studies were published in 2015 [6] [7]. The technique is promising, but there are some major challenges to solve before SRUS can be implemented clinically [27]. Firstly, time is an important issue. In order to acquire a sufficient number of image frames that allow us to localize enough microbubbles to get a reliable and sufficient representation of the vasculature, each scan session will inevitably last from several seconds to minutes, depending on the area of interest, equipment, and scan technique [28]. Time is a challenge because the microscopic vascular structures move during the long acquisitions, especially in larger animals and humans. Several groups have been and are still working on ways to make scan time faster, including imaging overlying microbubbles [8], scanning without contrast agents [29], or using deep learning [30]. Basing vascular evaluations on microbubble trajectories is also uncertain, as the number of microbubbles can vary substantially between scans, naturally affecting the estimated parameters such as vessel density [31]. Another big hurdle is imaging detailed and complex vascular structures in 2 D. Not only is out-of-plane motion a challenge as it cannot be compensated for, but all the vessels that naturally wind and twine in the elevational direction of the ultrasound beam should be considered in the acquisition and interpretation of SRUS [32]. The out-of-plane-vessels are projected into 2 D in the SRUS images, which may result in underestimated velocities and incorrect estimations of vascular parameters such as tortuosity. Introduction of 3 D SRUS may alleviate some of these challenges [33] [34]. Lastly, SRUS allows imaging of very small blood vessels but not necessary at the capillary level nor even at the arterioler/venular level, depending for example on the vascular density and complexity. Therefore, defining the level of the vasculature that is possible to image and how to improve it is another important challenge.

Conclusively, with continuous improvements in scanning equipment and optimized processing algorithms, the next natural question is where and how SRUS will have a clinical impact. Research-wise, there are many unanswered questions to pursue. However, in the clinic, it will be interesting to see where SRUS can make a difference: In the person with diabetes whose renal vasculature is starting to get affected by the disease and early treatment initiation is critical, or in the crucial early evaluation of advanced cancer treatment effect as an add-on to the normally-used RESIST criteria?

Sofie Bech Andersen

Charlotte Mehlin Sørensen

Jørgen Arendt Jensen

Michael Bachmann Nielsen

Conflict of Interest

The authors declare that they have no conflict of interest.

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Publication History

Article published online:
05 December 2022