Ultrasound localization microscopy

Abstract

Ultrasound Localization Microscopy (ULM) is an emerging technique that provides impressive super-resolved images of microvasculature, i.e., images with much better resolution than the conventional diffraction-limited ultrasound techniques and is already taking its first steps from preclinical to clinical applications. In comparison to the established perfusion or flow measurement methods, namely contrast-enhanced ultrasound (CEUS) and Doppler techniques, ULM allows imaging and flow measurements even down to the capillary level. As ULM can be realized as a post-processing method, conventional ultrasound systems can be used for. ULM relies on the localization of single microbubbles (MB) of commercial, clinically approved contrast agents. In general, these very small and strong scatterers with typical radii of 1-3 µm are imaged much larger in ultrasound images than they actually are due to the point spread function of the imaging system. However, by applying appropriate methods, these MBs can be localized with sub-pixel precision. Then, by tracking MBs over successive frames of image sequences, not only the morphology of vascular trees but also functional information such as flow velocities or directions can be obtained and visualized. In addition, quantitative parameters can be derived to describe pathological and physiological changes in the microvasculature. In this review, the general concept of ULM and conditions for its applicability to microvessel imaging are explained. Based on this, various aspects of the different processing steps for a concrete implementation are discussed. The trade-off between complete reconstruction of the microvasculature and the necessary measurement time as well as the implementation in 3D are reviewed in more detail, as they are the focus of current research. Through an overview of potential or already realized preclinical and clinical applications - pathologic angiogenesis or degeneration of vessels, physiological angiogenesis, or the general understanding of organ or tissue function - the great potential of ULM is demonstrated.

Keywords: Blood flow velocities; Contrast agents; Localization microscopy; Microvasculature; Super-resolution; Tracking.

Figure 1

B-mode image of a murine xenograft tumor (a). Maximum intensity persistence image (b), ULM occurrences (c), ULM velocities (d), and ULM flow directions (e) in the tumor. Data were recorded at University Hospital Aachen with a Visualsonics Vevo 3100 at 18 MHz with transducer MX250S and MicroMarker contrast agent and processed with the ULM algorithms published in . Approval of the animal experiment by the German State Office for Nature, Environment and Consumer Protection (LANUV) North Rhine-Westphalia.

Figure 2

Ultrafast Doppler Imaging of the intact spine of a rat (a) and Ultrasound Localization Microscopy (b) in the same animal. Scale bar 1 mm for both images. Figure adapted from , creative commons license CC-BY-4.0, combined from Fig. 2A (intact) and Fig. 4B (intact) and scaled to identical scale.

Figure 3

Basic concept of ULM. (a-c) Point spread function (PSF) of an MB in a B-mode image. The PSF is much larger than the original size of the MB (a: the MB is magnified and shown as red dot) and often disrupted by noise (b). The sub-pixel position of the MB (b and c: red cross) can be localized by, e.g., fitting a bivariate Gaussian function (see c) to the PSF. (d) Schematic illustration of the localization of single MBs in frame 1 to 3 of an imaging sequence. (e) Maximum intensity projection (MIP) of frame 1 to 3 using contrast-enhanced ultrasound. (f) Tracking of the MB’s positions from frame to frame (exemplarily shown for frame 1 to 2) allows the reconstruction of vessel courses and the determination of flow velocities and directions. (g) Reconstruction of the vessel tree from the full imaging sequence.

Figure 4

ULM processing steps. The example shown is a preclinical sequence of a mouse brain recorded with a MX700 transducer (29-71 MHz) on a Vevo 3100 (both Fujifilm Visualsonics). The contrast agent used for the CEUS sequence was MicroMarker (Fujifilm Visualsonics, see Table 1).

Figure 5

Reconstruction of the microvasculature of an A431 murine xenograft tumor (data set 2, see Section IV results) after (a) 10, (b) 20, and (c) 40 s. From under Creative Commons License CC-BY-4.0.

Figure 6

a) 3D ULM of a mouse brain in coronal (i) sagittal (ii) and top (iii) views. b) The zoom shows resolution at the microvascular level with measurements of the vessel diameters. Adapted from under the creative commons license CC-BY-4.0.

Figure 7

Estimation of the final percentage of pixels containing vessels (${\widehat{P}}_v$, blue) in dependence of the number of frames used for the estimation. For comparison, the percentage of pixels containing vessels in the current frame ($C$, green) is shown. Adapted from Fig. 5a showing only the relevant curves under the creative commons license CC-BY-4.0.