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Ultrasound localization microscopy can map blood vessels with a resolution much smaller than the wavelength by localizing microbubbles. The current implementations of the technique are limited to 2-D planes or small fields of view in 3-D. These suffer from minute-long acquisitions, out-of-plane microbubbles, and tissue motion. In this paper, we exploit the recent development of 4D ultrafast ultrasound imaging to insonify an isotropic volume up to 20 000 times per second and perform localization microscopy in the three dimensions. Specifically, a 32 ×32 elements, 9-MHz matrix-array probe connected to a 1024-channel programmable ultrasound scanner was used to achieve sub-wavelength volumetric imaging of both the structure and vector flow of a complex 3D structure (a main canal branching out into two side canals). To cope with the large volumes and the need to localize the bubbles in the three dimensions, novel algorithms were developed based on deconvolution of the beamformed microbubble signal. For tracking, individual particles were paired following a Munkres assignment method, and velocimetry was done following a Lagrangian approach. ULM was able to clearly represent the 3-D shape of the structure with a sharp delineation of canal edges (as small as [Formula: see text]) and separate them with a spacing as low as [Formula: see text]. The compounded volume rate of 500 Hz was sufficient to describe velocities in 2.5-150-mm/s range and to reduce the maximum acquisition time to 12 s. This paper demonstrates the feasibility of in vitro 3-D ultrafast ultrasound localization microscopy and opens up the way toward in vivo volumetric ULM.

Ultrafast 3D Ultrasound Localization Microscopy Using a 32 $\times$ 32 Matrix Array