he lymphatic system plays an important role in human circulatory and immune homeostasis. Peripheral lymph nodes (LNs) are regional hubs that receive interstitial fluid as well as circulating blood. They contain large numbers of immune cells and the complex internal architecture of LNs constitutes an interface between the cellular and molecular constituents of lymph and blood. Cellular debris is filtered and primary immune responses can be generated in LNs to pathogens (1). LN blood vessels are highly specialized for facilitating T-cell entry to the node (2). The spatial distribution of surface area for exchange of fluid with blood vessels in LNs determines the amount of local fluid movement, and it is therefore important to quantify the three-dimensional distribution of LN blood vessels. In patients with cancer, tumor cells can also traffic through lymphatic channels to regional LNs (3). Recent studies have demonstrated that metastatic cells migrate toward and into LN blood vessels (4,5). Malignant processes are associated with both angiogenesis and lymphangiogenesis (6). LNs may even act as a permissive niche to support the proliferation of metastatic cells and promote movement to distant sites (7). The accurate identification and quantification of metastatic LNs remains essential for prognosis and treatment planning (8).

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 superresolution 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 7millisecond 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 superresolution imaging and super-resolved velocity mapping using a customized 2D sparse array transducer.

Localization-based ultrasound super-resolution imaging using microbubble contrast agents and phase-change nanodroplets 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 superresolved frames with subsecond 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 were investigated. The experimental results show that subwavelength microstructures as small as 190 µm 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 the conventional B-mode, and two orders of magnitude faster than the recently reported AWSALM under a nonflow/very slow flow situations and other localization-based methods. Just as in AWSALM, fast-AWSALM does not require flow, as is required by current microbubblebased 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 milliseconds and does not require flow.

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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 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 /10 to /20 while imaging the microvasculature structure modelled 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 a 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.