Accurate volumetric velocity estimation is crucial in ultrasound imaging for both diagnostic and therapeutic applications. Traditional ultrasound systems, though effective for two-dimensional imaging, face major limitations in 3D imaging due to hardware and computational demands. Row-column addressed (RCA) ultrasound probes offer a promising alternative by reducing hardware complexity, thereby reducing the gap between research prototypes and clinical systems. However, this typically comes at the expense of stronger sidelobes compared with fully populated matrix arrays, leading to reduced image contrast. Several approaches have been proposed to improve the contrast of power Doppler imaging, yet the accuracy and performance of velocity Doppler estimation have received comparatively little attention. In this study, we present a method that exploits the phase information from RCA row and column signals to derive a novel velocity estimator based on cross-correlation of orthogonal apertures. This extends the XDoppler scheme, initially developed for power Doppler imaging, to velocity estimation. The XDoppler estimator is shown to provide accurate measurements of axial velocities and to outperform the traditional phase-shift autocorrelator, while offering a theoretical Nyquist velocity twice as high. In vitro experiments further demonstrate enhanced sensitivity to slow flows and reduced bias in flow rate estimation. In vivo data from a carotid artery confirm the reduced sensitivity to aliasing and reveal the ability to track dynamic blood flow velocity changes associated with arterial pulsatility. These findings suggest that the XDoppler velocity estimator could improve volumetric velocity imaging in clinical contexts.

Functional ultrasound imaging (fUSI) is a promising tool for studying brain activity in awake and behaving animals, offering insights into neural dynamics that are more naturalistic than those obtained under anesthesia. However, motion artifacts pose a significant challenge, introducing biases that can compromise the integrity of the data. This study provides a comprehensive evaluation and benchmarking of strategies for detecting and removing motion artifacts in transcranial fUSI acquisitions of awake mice. We evaluated 792 denoising strategies across four datasets, focusing on clutter filtering, scrubbing, frequency filtering, and confound regression methods. Our findings highlight the superior performance of adaptive clutter filtering and aCompCor confound regression in mitigating motion artifacts while preserving functional connectivity patterns. We also demonstrate that high-pass filtering is generally more effective than band-pass filtering in the presence of motion artifacts. Additionally, we show that with effective clutter filtering, scrubbing may become optional, which is particularly beneficial for experimental designs where motion correlates with conditions of interest. Based on these insights, we propose two optimized denoising paradigms tailored to different experimental constraints, providing practical recommendations for enhancing the reliability and reproducibility of fUSI data. Our findings challenge current practices in the field and have immediate practical implications for existing fUSI analysis workflows, paving the way for more sophisticated applications of fUSI in studying complex brain functions and dysfunctions in awake experimental paradigms.

Neuropathic pain arises from complex peripheral and central mechanisms and induces long-lasting maladaptive changes in the brain. To investigate the temporal dynamics of these changes, we examined resting-state functional connectivity (FC) in a mouse model of neuropathic pain across its initiation and maintenance phases. Using functional ultrasound imaging to capture whole-brain FC over disease progression, we conducted two complementary studies: a longitudinal assessment in anesthetized animals and an analysis of awake cohorts at distinct disease stages. Both exploratory and literature-guided analyses revealed that FC across most large-scale networks remained remarkably stable during pain maintenance. In contrast, pain onset was marked by enhanced FC between key regions involved in sensory, emotional, and motivational processing, including the motor cortex and nucleus accumbens, the prelimbic and insular cortices, and the infralimbic cortex and hypothalamus. As pain persisted, we observed reduced FC within the somatomotor network, reflecting functional disconnection. Additionally, postsurgical pain alone produced enduring FC changes within the prefrontal cortex, hippocampus, and insula, indicating long-term central plasticity independent of neuropathic injury. Together, these findings reveal dynamic, network-specific adaptations that distinguish the onset and maintenance phases of neuropathic pain and surgery-related plasticity. ### Competing Interest Statement MT, TD and BFO are co-founders and shareholders of Iconeus company. MT and TD are co-inventor of several patents in the field of neurofunctional ultrasound and ultrafast ultrasound. MT and TD do not have any other financial conflict of interest, nor any non-financial conflict of interests. SLMED, LE, AB and JF are currently employed by Iconeus. SC's PhD was partially funded by Iconeus. All the other authors do not have any financial or non-financial conflict of interests. Agence Nationale de la Recherche, Project ‘PINCH’, 18-CE37-0005-01

Brain perfusion relies on a complex vascular network of arteries, veins, and capillaries to meet its constant demand for oxygen and nutrients. Disruption of this microvascular system is a hallmark of many neurological disorders, including small vessel disease, stroke, and brain tumors. As such, high-resolution in vivo imaging of cerebral microvascular flow and structure remains critical to understanding these pathologies. Among them, ultrasound localization microscopy (ULM) allows noninvasive imaging of microvascular network down to small arterioles and venules at subwavelength resolution using injected microbubbles, but the approach remains mainly limited to 2D imaging with few volumetric implementations. In this study, we explore in vivo transcranial 3D ULM of the mouse brain using row-column arrays (RCA) and introduce an analysis framework to build a flow-directed vascular graph from the ULM microbubble tracking data, allowing to differentiate between subgraphs of artery-like and vein-like vascular segments. Using this framework, we are able to quantify flow and radius relationships for each subgraph in different anatomical regions. This high-sensitivity framework enables in vivo microvascular imaging and quantification in mice and provides a scalable platform for preclinical neurovascular studies in health and disease.

Ultrasound Localization Microscopy (ULM) is a super-resolution imaging technique that enables in vivo visualization of the brain's microvascular architecture surpassing the diffraction limit of conventional ultrasound. By detecting and tracking intravenously injected microbubbles as they circulate through cerebral vessels, ULM produces high-resolution maps of vascular density, flow velocity, and backscattered signal amplitude at spatial scales down to 5-10 µm. This protocol presents a complete workflow for performing ULM imaging in rodents, including animal preparation, probe positioning, image acquisition, microbubble injection, and data processing, using a dedicated functional ultrasound platform. Two preparation methods are described, adapted for mice (transcranial) and rats (with cranial windows), followed by detailed instructions for probe alignment and anatomical targeting using an integrated brain atlas. During acquisition, ultrafast ultrasound sequences are synchronized with bolus injections of microbubbles to capture dynamic flow data. Subsequent reconstruction steps involve clutter filtering, image interpolation, microbubble detection, subpixel localization, and trajectory tracking. Outputs include density maps reflecting vessel occupancy, velocity maps revealing flow patterns and directionality, and amplitude maps offering additional contrast for structural interpretation. Representative results illustrate successful acquisition across full coronal planes and highlight common pitfalls such as poor injection quality, motion artifacts, and skull-induced aberration. The protocol is compatible with both cross-sectional and longitudinal studies and is particularly suited for investigating cerebrovascular alterations in models of aging, stroke, aneurysm, and neurodegenerative diseases. By combining depth penetration, high spatiotemporal resolution, and label-free vascular imaging, ULM offers a powerful tool for noninvasive brain microcirculation analysis in preclinical models.

Functional ultrasound imaging (fUS) and ultrasound localization microscopy (ULM) are advanced ultrasound imaging modalities for assessing both functional and anatomical characteristics of the brain. However, the application of these techniques at a whole-brain scale has been limited by technological challenges. While conventional linear acoustic probes provide a narrow 2D field of view and matrix probes lack sufficient sensitivity for 3D transcranial fUS, multi-array probes have been developed to combine high sensitivity to blood flow with fast 3D acquisitions. In this study, we present a novel approach for the combined implementation of transcranial whole-brain fUS and ULM in mice using a motorized multi-array probe. This technique provides high-resolution, non-invasive imaging of neurovascular dynamics across the entire brain. Our findings reveal a significant correlation between absolute cerebral blood volume (ΔCBV) increases and microbubble speed, indicating vessel-level dependency of the evoked response. However, the lack of correlation with relative CBV (rCBV) suggests that fUS cannot distinguish functional responses alterations across different arterial vascular compartments. This methodology holds promise for advancing our understanding of neurovascular coupling and could be applied in brain disease diagnostics and therapeutic monitoring.

The quest to decode the complex supraspinal mechanisms that integrate cutaneous thermal information in the central system is still ongoing. The dorsal horn of the spinal cord is the first hub that encodes thermal input which is then transmitted to brain regions via the spinothalamic and thalamocortical pathways. So far, our knowledge about the strength of the interplay between the brain regions during thermal processing is limited. To address this question, we imaged the brains of adult awake male mice in resting state using functional ultrasound imaging during plantar exposure to constant and varying temperatures. Our study reveals for the first time the following: (1) a dichotomy in the response of the somatomotor-cingulate cortices and the hypothalamus, which was never described before, due to the lack of appropriate tools to study such regions with both good spatial and temporal resolutions. (2) We infer that cingulate areas may be involved in the affective responses to temperature changes. (3) Colder temperatures (ramped down) reinforce the disconnection between the somatomotor-cingulate and hypothalamus networks. (4) Finally, we also confirm the existence in the mouse brain of a brain mode characterized by low cognitive strength present more frequently at resting neutral temperature. The present study points toward the existence of a common hub between somatomotor and cingulate regions, whereas hypothalamus functions are related to a secondary network.

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet-unknown functions. We applied laser speckle imaging, ultrafast ultrasound, and two-photon microscopy in a thrombin-based mouse model of stroke and fibrinolytic treatment to show that LMCs maintain cerebral autoregulation and allow for gradual reperfusion, resulting in small infarcts. In mice with poor LMCs, distal arterial segments collapse, and deleterious hyperemia causes hemorrhage and mortality after recanalization. In silico analyses confirm the relevance of LMCs for preserving perfusion in the ischemic region. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion and preventing futile recanalization after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for beneficial reperfusion after stroke.

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet unknown functions. Utilizing a thrombin-based mouse model of stroke and the gold standard fibrinolytic treatment rt-PA, we here show that LMCs play a critical role in preserving vascular function in ischemic territories. We applied laser speckle contrast imaging, ultrafast ultrasound, and two-photon microscopy, to show that after thrombolysis, LMCs allow for gradual reperfusion resulting in small infarcts. On the contrary, in mice with poor LMCs, distal segments of recanalized arteries collapse and deleterious hyperemia causes hemorrhage and mortality. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for gradual reperfusion of ischemic tissues after stroke.

Functional ultrasound (fUS) has received growing attention in preclinical research in the past decade, providing a new tool to measure functional connectivity (FC) and brain task-evoked responses with single-trial detection capability in both anesthetized and awake conditions. Most fUS studies rely on 2D linear arrays to acquire one slice of the brain. Volumetric fUS using 2D matrix or row-column arrays has recently been demonstrated in rats and mice but requires invasive craniotomy to expose the brain due to a lack of sensitivity. In a previous study, we proposed the use of motorized linear arrays, allowing imaging through the skull in mice for multiple slices with high sensitivity. However, the tradeoff between the field of view and temporal resolution introduced by motorized scanning prevents acquiring brain-wide resting-state FC data with a sufficient volume rate for resting-state FC analysis. Here, we propose a new hybrid solution optimized and dedicated to brain-wide transcranial FC studies in mice, based on a newly developed multi-array transducer allowing simultaneous multi-slicing of the entire mouse cerebrum. We first demonstrate that our approach provides a better imaging quality compared to other existing methods. Then, we show the ability to image the whole mouse brain non-invasively through the intact skin and skull during visual stimulation under light anesthesia to validate this new approach. Significant activation was detected along the whole visual pathway, at both single and group levels, with more than 10% of augmentation of the cerebral blood volume (CBV) signal during the visual stimulation compared to baseline. Finally, we assessed resting-state FC in awake head-fixed animals. Several robust and long-ranged FC patterns were identified in both cortical and sub-cortical brain areas, corresponding to functional networks already described in previous fMRI studies. Together, these results show that the multi-array probe is a valuable approach to measure brain-wide hemodynamic activity in mice with an intact skull. Most importantly, its ability to identify robust resting-state networks is paving the way towards a better understanding of the mouse brain functional organization and its breakdown in genetic models of neuropsychiatric diseases.

Background Non-invasive high-resolution imaging of the cerebral vascular anatomy and function is key for the study of intracranial aneurysms, stenosis, arteriovenous malformations, and stroke, but also neurological pathologies, such as degenerative diseases. Direct visualization of the microvascular networks in the whole brain remains however challenging in vivo. Methods In this work, we performed 3D ultrafast ultrasound localization microscopy (ULM) using a 2D ultrasound matrix array and mapped the whole-brain microvasculature and flow at microscopic resolution in C57Bl6 mice in vivo. Findings We demonstrated that the mouse brain vasculature can be imaged directly through the intact skull at a spatial resolution of 20 mu m and over the whole brain depth and at high temporal resolution (750 volumes.s(-1)). Individual microbubbles were tracked to estimate the flow velocities that ranged from 2 mm.s(-1) in arterioles and venules up to 100 mm.s(-1) in large vessels. The vascular maps were registered automatically with the Allen atlas in order to extract quantitative vascular parameters such as local flow rates and velocities in regions of interest. Interpretation We show the potential of 3D ULM to provide new insights into whole-brain vascular flow in mice models at unprecedented vascular scale for an in vivo technique. This technology is highly translational and has the potential to become a major tool for the clinical investigation of the cerebral microcirculation. Funding This study was supported by the European Research Council under the European Union's Seventh Framework Program (FP/2007-2013) / ERC Grant Agreement n degrees 311025 and by the Fondation Bettencourt-Schueller under the program "Physics for Medicine". We acknowledge the ART (Technological Research Accelerator) biomedical ultrasound program of INSERM. Copyright (C) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

Row column addressing (RCA) transducers have the potential to provide volumetric imaging at ultrafast frame rate using a low channel count over a large field of view. In previous works we have shown that vascular imaging of large arteries as well as functional neuroimaging of the rat brain were feasible using RCA orthogonal plane wave imaging (OPW), but these applications required to transmit many plane waves, significantly reducing the frame rate. In this study, we introduce XDoppler imaging, a novel method to increase the performances of RCA flow imaging by taking advantage of the blood spatial decorrelation statistics combined with the limited spatial overlap of the point spread functions (PSF) of the two orthogonal apertures of the RCA transducer. We provide at first a theoretical basis to understand how the correlation operation reduces the sidelobe level. Then, we demonstrate both in vitro and in vivo in the human carotid artery and in the rat brain that XDoppler provides a significant gain in contrast-to-noise ratio (CNR) (between 3 and 6 dB depending on the application) compared to OPW. This improvement also leads to a sensitivity increase in the rat brain as more blood vessels are detected by XDoppler imaging.

There is a critical need for reliable quantitative biomarkers to assess functional brain alterations in mouse models of neuropsychiatric diseases, but current imaging methods measuring drug effects through the neurovascular coupling, face issues including poor sensitivity, drug-induced changes in global brain perfusion and the effects of anesthesia. Here we demonstrate the proof-of-concept of a minimally-invasive fUS imaging approach to detect the acute cholinergic modulatory effects of Scopolamine (ScoP) on functional brain connectivity in awake and behaving mice, through the intact skull. A machine-learning algorithm constructed an ad-hoc pharmacological score from the ScoP-induced changes in connectivity patterns of five mice. The discrimination model shows important ScoP-induced increase of the hippocampo-cortical connectivity. The pharmacological score led to robust discrimination of ScoP treatment from baseline in an independent dataset and showed, in another independent group, dose-dependent specific effects of central cholinergic modulation of functional connectivity, independent from global brain perfusion changes. In conclusion, we introduce pharmaco-fUS as a simple, robust, specific and sensitive modality to monitor drug effects on perfusion and functional connectivity in the awake mouse brain.