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Abstract Background Specific knowledge of the molecular pathways controlling host-pathogen interactions can increase our understanding of immune response biology as well as provide targets for drug development and genetic improvement of disease resistance. Toward this end, we have characterized the porcine transcriptional response to Salmonella enterica serovar Choleraesuis (S. Choleraesuis), a Salmonella serovar that predominately colonizes swine, yet can cause serious infections in human patients. Affymetrix technology was used to screen for differentially expressed genes in pig mesenteric lymph nodes (MLN) responding to infection with S. Choleraesuis at acute (8 hours (h), 24 h and 48 h post-inoculation (pi)) and chronic stages (21 days (d) pi). Results Analysis of variance with false discovery rate control identified 1,853 genes with significant changes in expression level (p-value < 0.01, q-value < 0.26, and fold change (FC) > 2) during infection as compared to un-inoculated control pigs. Down-regulation of translation-related genes at 8 hpi and 24 hpi implied that S. Choleraesuis repressed host protein translation. Genes involved in the Th1, innate immune/inflammation response and apoptosis pathways were induced significantly. However, antigen presentation/dendritic cell (DC) function pathways were not affected significantly during infection. A strong NFκB-dependent response was observed, as 58 known NFκB target genes were induced at 8, 24 and/or 48 hpi. Quantitative-PCR analyses confirmed the microarray data for 21 of 22 genes tested. Based on expression patterns, these target genes can be classified as an "Early" group (induced at either 8 or 24 hpi) and a "Late" group (induced only at 48 hpi). Cytokine activity or chemokine activity were enriched within the Early group genes GO annotations, while the Late group was predominantly composed of signal transduction and cell metabolism annotated genes. Regulatory motif analysis of the human orthologous promoters for both Early and Late genes revealed that 241 gene promoters were predicted to contain NFκB binding sites, and that of these, 51 Early and 145 Late genes were previously not known to be NFκB targets. Conclusion Our study provides novel genome-wide transcriptional profiling data on the porcine response to S. Choleraesuis and expands the understanding of NFκB signaling in response to Salmonella infection. Comparison of the magnitude and timing of porcine MLN transcriptional response to different Salmonella serovars, S. Choleraesuis and S. Typhimurium, clearly showed a larger but later transcriptional response to S. Choleraesuis. Both microarray and QPCR data provided evidence of a strong NFκB-dependent host transcriptional response during S. Choleraesuis infection. Our data indicate that a lack of strong DC-mediated antigen presentation in the MLN may cause S. Choleraesuis infected pigs to develop a systemic infection, and our analysis predicts nearly 200 novel NFκB target genes which may be applicable across mammalian species.

Glomeruli are the filtration units of the kidney and their function relies heavily on their microcirculation. Despite its obvious diagnostic importance, an accurate estimation of blood flow in the capillary bundle within glomeruli defies the resolution of conventional imaging modalities. Ultrasound Localization Microscopy (ULM) has demonstrated its ability to image in-vivo deep organs in the body. Recently, the concept of sensing ULM or sULM was introduced to classify individual microbubble behavior based on the expected physiological conditions at the micrometric scale. In the kidney of both rats and humans, it revealed glomerular structures in 2D but was severely limited by planar projection. In this work, we aim to extend sULM in 3D to image the whole organ and in order to perform an accurate characterization of the entire kidney structure. The extension of sULM into the 3D domain allows better localization and more robust tracking. The 3D metrics of velocity and pathway angular shift made glomerular mask possible. This approach facilitated the quantification of glomerular physiological parameter such as an interior traveled distance of approximately 7.5 ±0.6 microns within the glomerulus. This study introduces a technique that characterize the kidney physiology which can serve as a method to facilite pathology assessment. Furthermore, its potential for clinical relevance could serve as a bridge between research and practical application, leading to innovative diagnostics and improved patient care.

Super-resolution ultrasound (SRUS) has evolved significantly with the advent of Ultrasound Localization Microscopy (ULM). This technique enables sub-wavelength resolution imaging using microbubble contrast agents. Initially confined to 2D imaging, ULM has progressed towards volumetric approaches, allowing for comprehensive three-dimensional visualization of microvascular networks. This review explores the technological advancements and challenges associated with volumetric ULM, focusing on key aspects such as transducer design, acquisition speed, data processing algorithms, or integration into clinical practice. We discuss the limitations of traditional 2D ULM, including dependency on precise imaging plane selection and compromised resolution in microvasculature quantification. In contrast, volumetric ULM offers enhanced spatial resolution and allowed motion correction in all direction, promising transformative insights into microvascular pathophysiology. By examining current research and future directions, this review highlights the potential of volumetric ULM to contribute significantly to diagnostic across various medical conditions, including cancers, arteriosclerosis, strokes, diabetes, and neurodegenerative diseases.

In Ultrasound Localization Microscopy (ULM), achieving high-resolution images relies on the precise localization of contrast agent particles across a series of beamformed frames. However, our study uncovers an enormous potential: The process of delay-and-sum beamforming leads to an irreversible reduction of Radio-Frequency (RF) channel data, while its implications for localization remain largely unexplored. The rich contextual information embedded within RF wavefronts, including their hyperbolic shape and phase, offers great promise for guiding Deep Neural Networks (DNNs) in challenging localization scenarios. To fully exploit this data, we propose to directly localize scatterers in RF channel data. Our approach involves a custom super-resolution DNN using learned feature channel shuffling, non-maximum suppression, and a semi-global convolutional block for reliable and accurate wavefront localization. Additionally, we introduce a geometric point transformation that facilitates seamless mapping to the B-mode coordinate space. To understand the impact of beamforming on ULM, we validate the effectiveness of our method by conducting an extensive comparison with State-Of-The-Art (SOTA) techniques. We present the inaugural in vivo results from a wavefront-localizing DNN, highlighting its real-world practicality. Our findings show that RF-ULM bridges the domain shift between synthetic and real datasets, offering a considerable advantage in terms of precision and complexity. To enable the broader research community to benefit from our findings, our code and the associated SOTA methods are made available at https://github.com/hahnec/rf-ulm.

Objectives: Kidney diseases significantly impact individuals' quality of life and strongly reduce life expectancy. Glomeruli play a crucial role in kidney function. Current imaging techniques cannot visualize them due to their small size. Sensing ultrasound localization microscopy (sULM) has shown promising results for visualizing in vivo the glomeruli of human kidney grafts. This study aimed to evaluate the ability of sULM to visualize glomeruli in vivo in native human kidneys despite their depth and a shorter duration of ultrasound acquisition limited by the period of the patient's apnea. Sensing ultrasound localization microscopy parameters in native kidneys and kidney grafts and their consequence regarding glomeruli detection were also compared., Materials and Methods: Exploration by sULM was conducted in 15 patients with native kidneys and 5 with kidney allografts. Glomeruli were counted using a normalized distance metric projected onto sULM density maps. The difference in the acquisition time, the kidney depth, and the frame rate between native kidneys and kidney grafts and their consequence regarding glomeruli detection were assessed., Results: Glomerular visualization was achieved in 12 of 15 patients with native kidneys. It failed due to impossible breath-holding for 2 patients and a too-deep kidney for 1 patient. Sensing ultrasound localization microscopy found 16 glomeruli per square centimeter in the native kidneys (6-31) and 33 glomeruli per square centimeter in kidney transplant patients (18-55)., Conclusions: This study demonstrated that sULM can visualize glomeruli in native human kidneys in vivo. The proposed method may have many hypothetical applications, including biomarker development, assisting biopsy, or potentially avoiding it. It establishes a framework for improving the detection of local microstructural pathology, influencing the evaluation of allografts, and facilitating disease monitoring in the native kidney., Competing Interests: Conflicts of interest and sources of funding: O.C. holds patents in the field of ultrasound localization microscopy (EP4011299A1). O.C. is one founder and shareholder of the ResolveStroke startup and receives speaker fees from Bracco. J.M.C. receives speakers fees from Canon MS, General Electric MS, Hitachi MS, Philips Ultrasound, Siemens Ultrasound, and Supersonic Imagine. Other authors have no conflicts of interest to disclose. The authors also have disclosed that off-label use of SonoVue for the kidney is discussed in this article; readers should consult product labeling for approved indications., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

With the widespread interest and uptake of super-resolution ultrasound (SRUS) through localization and tracking of microbubbles, also known as ultrasound localization microscopy (ULM), many localization and tracking algorithms have been developed. ULM can image many centimeters into tissue in-vivo and track microvascular flow non-invasively with sub-diffraction resolution. In a significant community effort, we organized a challenge, Ultrasound Localization and TRacking Algorithms for Super-Resolution (ULTRA-SR). The aims of this paper are threefold: to describe the challenge organization, data generation, and winning algorithms; to present the metrics and methods for evaluating challenge entrants; and to report results and findings of the evaluation. Realistic ultrasound datasets containing microvascular flow for different clinical ultrasound frequencies were simulated, using vascular flow physics, acoustic field simulation and nonlinear bubble dynamics simulation. Based on these datasets, 38 submissions from 24 research groups were evaluated against ground truth using an evaluation framework with six metrics, three for localization and three for tracking. In-vivo mouse brain and human lymph node data were also provided, and performance assessed by an expert panel. Winning algorithms are described and discussed. The publicly available data with ground truth and the defined metrics for both localization and tracking present a valuable resource for researchers to benchmark algorithms and software, identify optimized methods/software for their data, and provide insight into the current limits of the field. In conclusion, Ultra-SR challenge has provided benchmarking data and tools as well as direct comparison and insights for a number of the state-of-the art localization and tracking algorithms.

Cerebral circulation ensures the proper functioning of the entire human body, and its interruption, i.e. stroke, leads to irreversible damage. However, tools for observing cerebral circulation are still lacking. Although MRI and CT scans serve as conventional methods, their accessibility remains a challenge, prompting exploration into alternative, portable, and non-ionizing imaging solutions like ultrasound with reduced costs. While Ultrasound Localization Microscopy (ULM) displays potential in high-resolution vessel imaging, its 2D constraints limit its emergency utility. This study delves into the feasibility of 3D ULM with multiplexed probe for transcranial vessel imaging in sheep brains, emulating human skull characteristics. Three sheep underwent 3D ULM imaging, compared with angiographic MRI, while skull characterization was conducted in vivo using ultrashort bone MRI sequences and ex vivo via micro CT. The study showcased 3D ULM's ability to highlight vessels, down to the Circle of Willis, yet within a confined 3D field-of-view. Future enhancements in signal, aberration correction, and human trials hold promise for a portable, volumetric, transcranial ultrasound angiography system.

Covalent adaptable networks and vitrimers are novel polymers with dynamic reversible bond exchange reactions for crosslinks, enabling them to modulate their properties between those of thermoplastics and thermosets. They have been gathering interest as materials for their recycling and self-healing properties. In this review, we discuss different molecular simulation efforts that have been used over the last decade to investigate and understand the nanoscale and molecular behaviors of covalent adaptable networks and vitrimers. In particular, molecular dynamics, Monte Carlo, and a hybrid of molecular dynamics and Monte Carlo approaches have been used to model the dynamic bond exchange reaction, which is the main mechanism of interest since it controls both the mechanical and rheological behaviors. The molecular simulation techniques presented yield sufficient results to investigate the structure and dynamics as well as the mechanical and rheological responses of such dynamic networks. The benefits of each method have been highlighted. The use of other tools such as theoretical models and machine learning has been included. We noticed, amongst the most prominent results, that stress relaxes as the bond exchange reaction happens, and that at temperatures higher than the glass transition temperature, the self-healing properties are better since more bond BERs are observed. The lifetime of dynamic covalent crosslinks follows, at moderate to high temperatures, an Arrhenius-like temperature dependence. We note the modeling of certain properties like the melt viscosity with glass transition temperature and the topology freezing transition temperature according to a behavior ruled by either the Williams-Landel-Ferry equation or the Arrhenius equation. Discrepancies between the behavior in dissociative and associative covalent adaptable networks are discussed. We conclude by stating which material parameters and atomistic factors, at the nanoscale, have not yet been taken into account and are lacking in the current literature.

Two-dimensional ultrasound transducers enable the acquisition of fully volumetric data that have been demonstrated to provide greater diagnostic information in the clinical setting and are a critical tool for emerging ultrasound methods, such as super-resolution and functional imaging. This technology, however, is not without its limitations. Due to increased fabrication complexity, some matrix probes with disjoint piezoelectric panels may require initial calibration. In this manuscript, two methods for calibrating the element positions of the Vermon 1024-channel 8 MHz matrix transducer are detailed. This calibration is a necessary step for acquiring high resolution B-mode images while minimizing transducer-based image degradation. This calibration is also necessary for eliminating vessel-doubling artifacts in super-resolution images and increasing the overall signal-to-noise ratio (SNR) of the image. Here, we show that the shape of the point spread function (PSF) can be significantly improved and PSF-doubling artifacts can be reduced by up to 10 dB via this simple calibration procedure., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: P.A.D. and G.F.P. declare inventorship on pending patents relevant to super-resolution microvascular imaging. O.C. holds patents in the field of ultrasound localization microscopy. O.C. is one founder and shareholder of the ResolveStroke startup.

Hypothesis: Calcium carbonate (CaCO 3 ) nanoparticles could have great potential for contrast-enhanced ultrasound imaging (CEUS) due to their gas-generating properties and sensitivity to physiological conditions. However, the use of nano CaCO 3 for biomedical applications requires the assistance of stabilizers to control the size and avoid the fast dissolution/recrystallization of the particles when exposed to aqueous conditions., Experiments: Herein, we report the stabilization of nano CaCO 3 using lignin, and synthesized core-shell amorphous CaCO 3 -lignin nanoparticles (LigCC NPs) with a diameter below 100 nm. We have then investigated the echogenicity of the LigCC NPs by monitoring the consequent generation of contrast in vitro for 90 min in linear and non-linear B-mode imaging., Findings: This research explores how lignin type and structure affect stabilization efficiency, lignin structuration around CaCO 3 cores, and particle echogenicity. Interestingly, by employing lignin as the stabilizer, it becomes possible to maintain the echogenic properties of CaCO 3 , whereas the use of lipid coatings prevents the production of signal generation in ultrasound imaging. This work opens new avenue for CEUS imaging of the vascular and extravascular space using CaCO 3 , as it highlights the potential to generate contrast for extended durations at physiological pH by utilizing the amorphous phase of CaCO 3 ., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J-S. Thomann has patent # LU102358B1 ; WO2022144136A1 pending to Luxembourg Institute of Science and Technology (LIST). L. Chuzeville has patent # LU102358B1;WO2022144136A1 pending to Luxembourg Institute of Science and Technology (LIST)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcranial ultrasound plays a limited role in neuroradiology due to its lack of resolution, planar imaging, and user-dependency. By breaching the diffraction limit using injected microbubbles, volumetric ultrasound localization microscopy (ULM) could help alleviate those issues. However, performing 3D ultrasound imaging at a high frame rate with sufficient signal-to-noise ratio to track individual microbubbles through the skull remains a challenge, especially with a portable scanner. In this study, we describe a ULM sequence suitable for volumetric transcranial imaging exploiting cylindrical emissions on multiplexed matrix probes, through simulations, hydrophone measurements, and flow phantoms. This geometry leads to a doubling of the peak acoustic pressure, up to 400 kPa, with respect to spherical emission and improved volume rate, up to 180 Hz. Cylindrical emissions also improve ULM saturation rate by 60% through a skull phantom. The assessment of microbubble velocity was also improved from 33% error in the average flow measured with spherical waves to a 5% error with cylindrical waves. Conversely, we demonstrate the detrimental impacts of cylindrical waves toward the field of view and isotropic sensitivity. Nevertheless, due to its enhanced signal-to-noise ratio and 3D nature, such a cylindrical volumetric sequence could be beneficial for ULM as a diagnostic tool in humans, especially when portability is a necessity.

In neurosurgery, a current challenge is to provide localized therapy in deep and difficult-to-access brain areas with millimeter accuracy. In this prospect, new surgical devices such as microrobots are being developed, which require controlled inbrain navigation to ensure the safety and efficiency of the intervention. In this context, the device tracking technology has to answer a three-sided challenge: invasiveness, performance, and facility of use. Although ultrasound seems appropriate for transcranial tracking, the skull remains an obstacle because of its significant acoustic perturbations. A compact and affordable ultrasound-based tracking system that minimizes skull-related disturbances is proposed here. This system consists of three emitters fixed on the patient's head and a one-millimeter receiver embedded in the surgical device. The 3D position of the receiver is obtained by trilateration based on time of flight measurements. The system demonstrates a submillimeter tracking accuracy through an 8.9 mm thick skull plate phantom. This result opens multiple perspectives in terms of millimeter accurate navigation for a large number of neurobiomedical devices.

Systemic injection of thrombolytic drugs is the gold standard treatment for non-invasive blood clot resolution. The most serious risks associated with the intravenous injection of tissue plasminogen activator-like proteins are the bleeding complication and the dose related neurotoxicity. Indeed, the drug has to be injected in high concentrations due to its short half-life, the presence of its natural blood inhibitor (PAI-1) and the fast hepatic clearance (0.9 mg/kg in humans, 10 mg/kg in mouse models). Overall, there is a serious need for a dose-reduced targeted treatment to overcome these issues. We present in this article a new acoustic cavitation-based method for polymer MBs synthesis, three times faster than current hydrodynamic-cavitation method. The generated MBs are ultrasound responsive, stable and biocompatible. Their functionalization enabled the efficient and targeted treatment of stroke, without side effects. The stabilizing shell of the MBs is composed of Poly-Isobutyl Cyanoacrylate (PIBCA), copolymerized with fucoidan. Widely studied for its targeting properties, fucoidan exhibit a nanomolar affinity for activated endothelium and activated platelets (P-selectins). Secondly, the thrombolytic agent (rtPA) was loaded onto microbubbles (MBs) with a simple adsorption protocol. Hence, the present study validated the in vivo efficiency of rtPA-loaded Fuco MBs to be over 50 % more efficient than regular free rtPA injection for stroke resolution. In addition, the relative injected rtPA grafted onto targeting MBs was 1/10th of the standard effective dose (1 mg/kg in mouse). As a result, no hemorrhagic event, BBB leakage nor unexpected tissue distribution were observed., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cedric CHAUVIERRE reports financial support was provided by French National Research Agency. Olivier COUTURE reports a relationship with Resolve Stroke company that includes: board membership. Cedric CHAUVIERRE has patent pending to INSERM Transfert. Louise FOURNIER has patent pending to INSERM Transfert., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Estimation of glomerular function is necessary to diagnose kidney diseases. However, the study of glomeruli in the clinic is currently done indirectly through urine and blood tests. A recent imaging technique called Ultrasound Localization Microscopy (ULM) has appeared. It is based on the ability to record continuous movements of individual microbubbles in the bloodstream. Although ULM improved the resolution of vascular imaging up to tenfold, the imaging of the smallest vessels had yet to be reported., Methods: We acquired ultrasound sequences from living humans and rats and then applied filters to divide the data set into slow-moving and fast-moving microbubbles. We performed a double tracking to highlight and characterize populations of microbubbles with singular behaviors. We decided to call this technique "sensing ULM" (sULM). We used post-mortem micro-CT for side-by-side confirmation in rats., Findings: In this study, we report the observation of microbubbles flowing in the glomeruli in living humans and rats. We present a set of analysis tools to extract quantitative information from individual microbubbles, such as remanence time or normalized distance., Interpretation: As glomeruli play a key role in kidney function, it would be possible that their observation yields a deeper understanding of the kidney. It could also be a tool to diagnose kidney diseases in patients. More generally, it will bring imaging capabilities closer to the functional units of organs, which is a key to understand most diseases, such as cancer, diabetes, or kidney failures., Funding: This study was funded by the European Research Council under the European Union Horizon H2020 program (ERC Consolidator grant agreement No 772786-ResolveStroke)., Competing Interests: Declaration of interests O.C. and V.H., hold patents in the field of ultrasound localization microscopy (EP4011299A1). O.C. and V.H. are founders and shareholders of the ResolveStroke startup. O.C. has received several grants: ERC Consolidator grant agreement no 772786-Resolve Stroke, ERC POC (2022–2023), Maturation SATT Lutech for ResolveStroke Startup, ANR FightClot, CNRS Prematuration for ResolveStroke Startup, RHU Quid NASH. O.C. gave a lecture at Bracco SAS and was an invited speaker at several conferences such as BMT Innsbruck. Other authors have no conflict of interest to disclose., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Chronic kidney disease is a major medical problem, causing more than a million deaths each year worldwide. Peripheral kidney microvascular damage characterizes most chronic kidney diseases, yet noninvasive and quantitative diagnostic tools to measure this are lacking. Ultrasound Localization Microscopy (ULM) can assess tissue microvasculature with unprecedented resolution. Here, we optimized methods on 35 kidney transplants and studied the feasibility of ULM in seven human kidney allografts with a standard low frame rate ultrasound scanner to access microvascular damage. Interlobar, arcuate, cortical radial vessels, and part of the medullary organization were visible on ULM density maps. The medullary vasa recta can be seen but are not as clear as the cortical vessels. Acquisition parameters were derived from Contrast-Enhanced Ultrasound examinations by increasing the duration of the recorded clip at the same plane. ULM images were compared with Color Doppler, Advanced Dynamic Flow, and Superb Microvascular Imaging with a contrast agent. Despite some additional limitations due to movement and saturation artifacts, ULM identified vessels two to four times thinner compared with Doppler modes. The mean ULM smallest analyzable vessel cross section was 0.3 ± 0.2 mm in the seven patients. Additionally, ULM was able to provide quantitative information on blood velocities in the cortical area. Thus, this proof-of-concept study has shown ULM to be a promising imaging technique for qualitative and quantitative microvascular assessment. Imaging native kidneys in patients with kidney diseases will be needed to identify their ULM biomarkers., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

We succeeded in freeze-drying monodisperse microbubbles without degrading their performance, that is, their monodispersity in size and echogenicity. We used microfluidic technology to generate cryoprotected highly monodisperse microbubbles (coefficient of variation [CV] <5%). By using a novel retrieval technique, we were able to freeze-dry the microbubbles and resuspend them without degradation, that is, keeping their size distribution narrow (CV <6%). Acoustic characterization performed in two geometries (a centimetric cell and a millichannel) revealed that the resuspended bubbles conserved the sharpness of the backscattered resonance peak, leading to CVs ranging between 5% and 10%, depending on the geometry. As currently observed with monodisperse bubbles, the peak amplitudes are one order of magnitude higher than those of commercial ultrasound contrast agents. Our work thus solves the question of storage and transportation of highly monodisperse bubbles. This work might open pathways toward novel clinical non-invasive measurements, such as local pressure, impossible to carry out with the existing commercial ultrasound contrast agents., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Objective: Ultrasound Localization Microscopy (ULM) provides images of the microcirculation in-depth in living tissue. However, its implementation in two-dimension is limited by the elevation projection and tedious plane-by-plane acquisition. Volumetric ULM alleviates these issues and can map the vasculature of entire organs in one acquisition with isotropic resolution. However, its optimal implementation requires many independent acquisition channels, leading to complex custom hardware., Methods: In this article, we implemented volumetric ultrasound imaging with a multiplexed 32 × 32 probe driven by a single commercial ultrasound scanner. We propose and compare three different sub-aperture multiplexing combinations for localization microscopy in silico and in vitro with a flow of microbubbles in a canal. Finally, we evaluate the approach for micro-angiography of the rat brain. The "light" combination allows a higher maximal volume rate than the "full" combination while maintaining the field of view and resolution., Results: In the rat brain, 100,000 volumes were acquired within 7 min with a dedicated ultrasound sequence and revealed vessels down to 31 μm in diameter with flows from 4.3 mm/s to 28.4 mm/s., Conclusion: This work demonstrates the ability to perform a complete angiography with unprecedented resolution in the living rat's brain with a simple and light setup through the intact skull., Significance: We foresee that it might contribute to democratize 3D ULM for both preclinical and clinical studies.

Ultrafast ultrasound localization microscopy can be used to detect the subwavelength acoustic scattering of intravenously injected microbubbles to obtain haemodynamic maps of the vasculature of animals and humans. The quality of the haemodynamic maps depends on signal-to-noise ratios and on the algorithms used for the localization of the microbubbles and the rendering of their trajectories. Here we report the results of benchmarking of the performance of seven microbubble-localization algorithms. We used metrics for localization errors, localization success rates, processing times and a measure of the reprojection of the localization of the microbubbles on the original beamformed grid. We combined eleven metrics into an overall score and tested the algorithms in three simulated microcirculation datasets, and in angiography datasets of the brain of a live rat after craniotomy, an excised rat kidney and a mammary tumour in a live mouse. The algorithms, metrics and datasets, which we have made openly available at https://github.com/AChavignon/PALA and https://doi.org/10.5281/zenodo.4343435 , will facilitate the identification or generation of optimal microbubble-localization algorithms for specific applications., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)