Ultrasound Localization Microscopy (ULM) is an ultrasound imaging technique that relies on the acoustic response of sub-wavelength ultrasound scatterers to map the microcirculation with an order of magnitude increase in resolution as compared to conventional ultrasound imaging.Initially demonstrated in vitro, ULM has matured and sees implementation in vivo for vascular imaging of organs or tumors in both animal models and humans. The performance of localization algorithms greatly defines the quality of vascular mapping. Here, we compiled and implemented a collection of ultrasound localization algorithms and devised three in silico and in vivo datasets to compare their performance through 11 metrics. We also present two novel algorithms designed to increase speed and overall performance. By providing a comprehensive open package to perform ULM that includes localization algorithms, the datasets used, and the evaluation metrics, we aim to equip researchers with a tool to identify the optimal localization algorithm for their 2 application, benchmark their own software and enhance the overall quality of their ULM images while uncovering the algorithms' own limits. MainThe circulatory system carries the essential nutrients of life to cells in the body. It forms a 100,000 kilometer-long network composed of centimeter-wide arteries down to capillaries that are a few micrometers in diameter at most. The study of the vascular system is essential for both the diagnosis and treatment of cardiovascular diseases, cancer, diabetes, stroke, or organ dysfunction.Due to its diversity of scale, imaging the vasculature is a daunting task and few techniques are capable of measuring micro-hemodynamics deep in the human body.Ultrasound imaging is extensively used in medical practice as a non-invasive tool that provides soft-tissue diagnosis, prognosis, or guides interventions. Using the Doppler effect, it can also measure blood flow in real-time. With the advent of plane wave techniques 1 , ultrasound has reached frame rates up to 20 kHz making it possible to observe and measure fast occurring changes 1 such as functional changes in the brain 2 , as well as increase sensitivity to blood flow that allows more accurate filtering 3,4 . To increase blood's contrast, micrometric gas microbubbles can be intravenously injected in vivo. Contrast-Enhanced Ultrasound (CEUS) is mostly used for perfusion studies and cardiac imaging 5 .Because conventional ultrasound imaging, Doppler, and contrast-enhanced ultrasound all rely on the propagation of sound waves, ultrasound imaging is largely limited in resolution by diffraction.Recently, Ultrasound Localization Microscopy (ULM) has broken that limit by isolating a small number of microbubbles as subwavelength sources in each image and localizing them with micrometric precision [6][7][8] . Similar to PALM (PhotoActivated Localization Microscopy), it uses

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The resolution of an imaging system is usually determined by the width of its point spread function and is measured using the Rayleigh criterion. For most system, it is in the order of the imaging wavelength. However, super resolution techniques such as localization microscopy in optical and ultrasound imaging can resolve features an order of magnitude finer than the wavelength. The classical description of spatial resolution no longer applies and new methods need to be developed.In optical localization microscopy, the Fourier Ring Correlation has showed to be an effective and practical way to estimate spatial resolution for Single Molecule Localization Microscopy data. In this work, we wish to investigate how this tool can provide a direct and universal estimation of spatial resolution in Ultrasound Localization Microscopy. Moreover, we discuss the concept of spatial sampling in Ultrasound Localization Microscopy and demonstrate how the Nyquist criterion for sampling drives the spatial/temporal resolution tradeoff.We measured spatial resolution on five different datasets over rodent's brain, kidney and tumor finding values between 11 m and 34 m for precision of localization between 11 m and 15 m. Eventually, we discuss from those in vivo datasets how spatial resolution in Ultrasound Localization Microscopy depends on both the localization precision and the total number of detected microbubbles.This study aims to offer a practical and theoretical framework for image resolution in Ultrasound Localization Microscopy.

Early diagnosis is a critical part of the emergency care of cerebral hemorrhages and ischemia. A rapid and accurate diagnosis of strokes reduces the delays to appropriate treatments and a better functional recovery. Currently, CTscan and MRI are the gold standards with constraints of accessibility, availability, and possibly some contraindications. The development of Ultrasound Localization Microscopy (ULM) has enabled new perspectives to conventional transcranial ultrasound imaging with increased sensitivity, penetration depth, and resolution. The possibility of volumetric imaging has increased the field-of-view and provided a more precise description of the microvascularisation. In this study, rats (n = 9) were subjected to thromboembolic ischemic stroke or intracerebral hemorrhages prior to volumetric ULM at the early phases after onsets. Although the volumetric ULM performed in the early phase of ischemic stroke revealed a large hypoperfused area in the cortical area of the occluded artery, it showed a more diffused hypoperfusion in the hemorrhagic model. Respective computations of a Microvascular Diffusion Index highlighted different patterns of perfusion loss during the first 24 h of these two strokes’ subtypes. Our study provides the first proof that this methodology should allow early discrimination between ischemic and hemorrhagic stroke with a potential toward diagnosis and monitoring in clinic.

Technologies to visualize whole organs across scales in vivo are essential for our understanding of biology in health and disease. To date, only post-mortem techniques achieve cellular resolution across entire organs. Here, we demonstrate in vivo volumetric ultrasound localization microscopy (ULM). We detail a universal methodological pipeline including dedicated 3D ULM, motion correction and realignment algorithms, as well as post-processing quantification of cerebral blood diameter and flow. We illustrate the power of this approach, by revealing the whole rat brain vasculature at a 14-fold improved resolution of 12 μm, and cerebral blood flows ranging from 1 to 120 mm/s. The exposed methodology and results pave the way to the investigation of in vivo vascular and hemodynamic processes across the mammalian brain in health and disease.INDEX TERMS ultrasound superresolution, 3D ultrasound imaging, neurovascular imaging rodent brain atlas

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Technologies to visualize whole organs across scales in vivo are essential for our understanding of biology in health and disease. To date, only post-mortem techniques such as perfused computed tomography scanning or optical microscopy of cleared tissues achieve cellular resolution across entire organs and imaging methods with equal performance in living mammalian organs have yet to be developed. Recently, 2D ultrasound localization microscopy has successfully mapped the fine-scale vasculature of various organs down to a 10 μm precision. However, reprojection issues and out-of-plane motion prevent complex blood flow quantification and fast volumetric imaging of whole organs. Here, we demonstrate for the first time in vivo volumetric ultrasound localization microscopy mapping of the rodent brain vasculature. We developed a complete methodological pipeline that includes specific surgery, a dedicated 3D ultrasound acquisition sequence, localization and tracking algorithms, motion correction and realignment, as well as the post-processing quantification of cerebral blood flow. We illustrate the power of this approach, by mapping the whole rat brain vasculature at a resolution of 12 μm, revealing mesoscopic to macroscopic vascular architectures and cerebral blood flows ranging from 1 to 100 mm/s. Our results pave the way to the investigation of in vivo vascular processes across the mammalian brain in health and disease, in a wide range of contexts and models.