Name: Ultrasound Localization Microscopy for Super-Resolution Mapping of the Rodent Brain Microvasculature (Video) | JoVE Journal
Uploaded: 2025-11-14T13:30:00.000+00:00
Duration: 10 min 36 s
Description: Learn Ultrasound Localization Microscopy for super-resolution mapping of rodent brain microvasculature. This protocol enables detailed visualization of vascular architecture and flow dynamics in vivo.

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5 minutes

The authors would like to thank Philippe Mateo and Lantonirina Abdoul-Agige at Physics for Medicine Paris for their support with animal handling and care. Figure 1 and Figure 2 were created with BioRender.com.

<ol><li>Jamadar, S. D., Behler, A., Deery, H., Breakspear, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+metabolic+costs+of+cognition%5BTitle%5D)+AND+%22Trends+Cogn+Sci%22%5BJournal%5D)">The metabolic costs of cognition.</a> Trends Cogn Sci. 29 (6), 541-555 (2025).</li>
<li>Renaudin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+ultrasound+localization+microscopy+reveals+brain-wide+neurovascular+activity+on+a+microscopic+scale%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Functional ultrasound localization microscopy reveals brain-wide neurovascular activity on a microscopic scale.</a> Nat Methods. 19 (8), 1004-1012 (2022).</li>
<li>Agarwal, N., Carare, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebral+Vessels%3A+An+overview+of+anatomy%2C+physiology%2C+and+role+in+the+drainage+of+fluids+and+solutes%5BTitle%5D)+AND+%22Front+Neurol%22%5BJournal%5D)">Cerebral Vessels: An overview of anatomy, physiology, and role in the drainage of fluids and solutes.</a> Front Neurol. 11, 611485(2021).</li>
<li>Ahmad, A., Patel, V., Xiao, J., Khan, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+neurovascular+system+in+neurodegenerative+diseases%5BTitle%5D)+AND+%22Mol+Neurobiol%22%5BJournal%5D)">The role of neurovascular system in neurodegenerative diseases.</a> Mol Neurobiol. 57 (11), 4373-4393 (2020).</li>
<li>Sweeney, M. D., Kisler, K., Montagne, A., Toga, A. W., Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+brain+vasculature+in+neurodegenerative+disorders%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">The role of brain vasculature in neurodegenerative disorders.</a> Nat Neurosci. 21 (10), 1318-1331 (2018).</li>
<li>Kolinko, Y., Krakorova, K., Cendelin, J., Tonar, Z., Kralickova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microcirculation+of+the+brain%3A+Morphological+assessment+in+degenerative+diseases+and+restoration+processes%5BTitle%5D)+AND+%22Rev+Neurosci%22%5BJournal%5D)">Microcirculation of the brain: Morphological assessment in degenerative diseases and restoration processes.</a> Rev Neurosci. 26 (1), 75-93 (2015).</li>
<li>Deffieux, T., Demene, C., Pernot, M., Tanter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+ultrasound+neuroimaging%3A+A+review+of+the+preclinical+and+clinical+state+of+the+art%5BTitle%5D)+AND+%22Curr+Opin+in+Neurobiol%22%5BJournal%5D)">Functional ultrasound neuroimaging: A review of the preclinical and clinical state of the art.</a> Curr Opin in Neurobiol. 50, 128-135 (2018).</li>
<li>Mace, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+ultrasound+imaging+of+the+brain%3A+Theory+and+basic+principles%5BTitle%5D)+AND+%22IEEE+Trans+Ultrason+Ferroelectr+Freq+Control%22%5BJournal%5D)">Functional ultrasound imaging of the brain: Theory and basic principles.</a> IEEE Trans Ultrason Ferroelectr Freq Control. 60 (3), 492-506 (2013).</li>
<li>Tanter, M., Fink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrafast+imaging+in+biomedical+ultrasound%5BTitle%5D)+AND+%22IEEE+Trans+Ultrason+Ferroelectr+Freq+Control%22%5BJournal%5D)">Ultrafast imaging in biomedical ultrasound.</a> IEEE Trans Ultrason Ferroelectr Freq Control. 61 (1), 102-119 (2014).</li>
<li>Errico, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrafast+ultrasound+localization+microscopy+for+deep+super-resolution+vascular+imaging%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging.</a> Nature. 527 (7579), 499-502 (2015).</li>
<li>Réaux-Le-Goazigo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasound+localization+microscopy+and+functional+ultrasound+imaging+reveal+atypical+features+of+the+trigeminal+ganglion+vasculature%5BTitle%5D)+AND+%22Commun+Biol%22%5BJournal%5D)">Ultrasound localization microscopy and functional ultrasound imaging reveal atypical features of the trigeminal ganglion vasculature.</a> Commun Biol. 5 (1), 330(2022).</li>
<li>Siepmann, M., Schmitz, G., Bzyl, J., Palmowski, M., Kiessling, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+tumor+vascularity+by+tracing+single+microbubbles%5BTitle%5D)+AND+%22IEEE+Int+Ultrason+Symp%22%5BJournal%5D)">Imaging tumor vascularity by tracing single microbubbles.</a> IEEE Int Ultrason Symp. , 1906-1908 (2011).</li>
<li>Couture, O., Hingot, V., Heiles, B., Muleki-Seya, P., Tanter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasound+localization+microscopy+and+super-resolution%3A+A+state+of+the+art%5BTitle%5D)+AND+%22IEEE+Trans+Ultrason+Ferroelectr+Freq+Control%22%5BJournal%5D)">Ultrasound localization microscopy and super-resolution: A state of the art.</a> IEEE Trans Ultrason Ferroelectr Freq Control. 65 (8), 1304-1320 (2018).</li>
<li>Dencks, S., Schmitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasound+localization+microscopy%5BTitle%5D)+AND+%22Z+Med+Phys%22%5BJournal%5D)">Ultrasound localization microscopy.</a> Z Med Phys. 33 (3), 292-308 (2023).</li>
<li>Renaudin, N., Pezet, S., Ialy-Radio, N., Demene, C., Tanter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Backscattering+amplitude+in+ultrasound+localization+microscopy%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Backscattering amplitude in ultrasound localization microscopy.</a> Sci Rep. 13 (1), 11477(2023).</li>
<li>Blinder, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cortical+angiome%3A+An+interconnected+vascular+network+with+noncolumnar+patterns+of+blood+flow%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">The cortical angiome: An interconnected vascular network with noncolumnar patterns of blood flow.</a> Nat Neurosci. 16 (7), 889-897 (2013).</li>
<li>van Raaij, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+blood+flow+and+volume+in+arterioles+and+venules+of+the+rat+cerebral+cortex+using+functional+micro-ultrasound%5BTitle%5D)+AND+%22Neuroimage%22%5BJournal%5D)">Quantification of blood flow and volume in arterioles and venules of the rat cerebral cortex using functional micro-ultrasound.</a> Neuroimage. 63 (3), 1030-1037 (2012).</li>
<li>Lindner, J., Song, J., Jayaweera, A. R., Sklenar, J., Kaul, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microvascular+rheology+of+definity+microbubbles+after+intra-arterial+and+intravenous+administration%5BTitle%5D)+AND+%22J+Am+Soc+Echocardiogr%22%5BJournal%5D)">Microvascular rheology of definity microbubbles after intra-arterial and intravenous administration.</a> J Am Soc Echocardiogr. 15 (5), 396-403 (2002).</li>
<li>Nouhoum, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+functional+ultrasound+brain+GPS+for+automatic+vascular-based+neuronavigation%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A functional ultrasound brain GPS for automatic vascular-based neuronavigation.</a> Sci Rep. 11, 15197(2021).</li>
<li>Lowerison, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-resolution+ultrasound+reveals+cerebrovascular+impairment+in+a+mouse+model+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22J+Endocrinol%22%5BJournal%5D)">Super-resolution ultrasound reveals cerebrovascular impairment in a mouse model of Alzheimer's disease.</a> J Endocrinol. 44 (9), 1-14 (2024).</li>
<li>Nyúl-Tóth, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+intravital+approaches+to+quantify+deep+vascular+structure+and+perfusion+in+the+aging+mouse+brain+using+ultrasound+localization+microscopy+%28ULM%29%5BTitle%5D)+AND+%22J+Cereb+Blood+Flow+Metab%22%5BJournal%5D)">Novel intravital approaches to quantify deep vascular structure and perfusion in the aging mouse brain using ultrasound localization microscopy (ULM).</a> J Cereb Blood Flow Metab. 0 (0), 1-19 (2024).</li>
<li>Demeulenaere, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+whole+brain+microvascular+imaging+in+mice+using+transcranial+3D+Ultrasound+Localization+Microscopy%5BTitle%5D)+AND+%22EBioMedicine%22%5BJournal%5D)">In vivo whole brain microvascular imaging in mice using transcranial 3D Ultrasound Localization Microscopy.</a> EBioMedicine. 79 (1), 103995(2022).</li>
</ol>

TD is co-founder and scientific advisor for Iconeus, which commercializes ultrasound neuroimaging scanners. PP, SR, AB, TM, MN, and JF are employees of Iconeus. All other authors declare no competing interests.

Ultrasound Localization Microscopy (ULM) represents a transformative advancement in ultrasound imaging, enabling super-resolution visualization of the microvasculature beyond the conventional diffraction limit. This discussion highlights the critical steps in the ULM protocol, addresses potential modifications and troubleshooting strategies, explores the limitations of the method, evaluates its significance compared to alternative imaging modalities, and considers its importance and potential applications in biomedical research.
Critical steps in the ULM protocol 
Several steps are crucial to the successful implementation of ULM, and their influence is illustrated in Figure 7. One of the most important steps is the administration of microbubbles, as both the concentration and delivery quality directly affect imaging outcomes. An optimal concentration is necessary to balance spatial resolution and detectability. Inadequate delivery, such as poor catheter placement or extravasation, can result in very low vascular signal and incomplete reconstructions, as shown in Figure 7B, where only a sparse number of microbubbles reach the bloodstream. High-frame-rate imaging, typically in the kilohertz range, is essential for capturing the rapid movement of microbubbles and enabling reliable tracking across frames. Any instability during the acquisition, such as motion introduced by breathing, poor fixation, or insufficient anesthesia, can degrade the localization accuracy. An example of this is illustrated in Figure 7C, where excessive motion during the acquisition results in a blurred and unusable density map.
Microbubble localization and tracking depend on advanced image processing algorithms that detect individual bubbles, perform subpixel localization, and reconstruct trajectories with sub-diffraction precision. Even with proper acquisition and processing, anatomical factors such as skull-induced aberrations can still affect image quality. In transcranial imaging, particularly in older mice, skull thickening can introduce acoustic distortion. This leads to characteristic shadowing artifacts beneath the sagittal suture, where no microbubbles are detected, as depicted in Figure 7D. Such aberrations reduce signal-to-noise ratio and can obscure vascular structures unless corrected with adaptive filtering or by refining probe placement.
When all steps are performed successfully, high-quality ULM maps are achievable, as shown in Figure 7A, where a dense and well-resolved vascular network is reconstructed through a transcranial approach.
<img alt="Brain imaging; fluorescence microscopy; four conditions: reference, extravasation, motion, aberration." class="xfigimg" src="/files/ftp_upload/68612/68612fig7.jpg" data-altupdatedat="1778558422000" data-alt="Figure 7"> 
Figure 7: Reference and troubleshooting examples for ULM imaging quality in mice. Panel (A) shows a high-quality transcranial ULM microbubble (MB) density map acquired in a young adult mouse, illustrating successful vascular mapping across the full coronal plane. Panel (B) displays an example of a poor vascular signal due to microbubble extravasation or insufficient venous access, resulting in sparse MB detections. Panel (C) shows an acquisition affected by excessive animal motion, producing a blurry and non-interpretable density map. Panel (D) highlights skull-induced acoustic aberration commonly observed in older mice, visible as a shadow cone beneath the sagittal suture where no MBs are detected due to reduced signal-to-noise ratio. These examples serve as visual references for identifying and addressing common experimental artifacts in ULM imaging. Scale bar: 1mm. <a href="https://www.jove.com/files/ftp_upload/68612/68612fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Limitations of the method
Despite its many advantages, ULM has inherent limitations that affect its applicability. Out-of-plane vessel bias is a significant issue in 2D imaging, as vessels extending outside the imaging plane are not adequately reconstructed, leading to incomplete vascular maps. This limitation can be mitigated by transitioning to volumetric (3D) imaging approaches22, although this increases computational complexity. Intravenous catheterization challenges also pose a hurdle, especially in small animal models, where achieving consistent microbubble delivery can be technically demanding. Microbubble stability is also a limiting factor, especially during extended imaging sessions. Due to their short circulation half-life, microbubbles may require repeated bolus injections or continuous infusion protocols to maintain adequate contrast throughout the acquisition period. Motion artifacts remain a significant concern, particularly for awake subjects, where even small involuntary movements can degrade super-resolution imaging. Finally, depth penetration in ULM is inherently limited by ultrasound attenuation. While this is generally not problematic for rodent imaging at high frequencies (e.g., 15 MHz), it becomes more challenging in larger animals such as swine or non-human primates. In these cases, lower-frequency probes may be required to achieve sufficient imaging depth, but this comes at the cost of reduced spatial resolution-underscoring a key trade-off between depth and detail that must be considered when translating ULM to larger preclinical models.
Significance of ULM compared to existing methods
Ultrasound Localization Microscopy (ULM) offers substantial advantages over conventional imaging modalities, particularly in the context of microvascular imaging. Among its most notable strengths is its ability to surpass the diffraction limit of traditional ultrasound, achieving sub-diffraction imaging at spatial scales comparable to optical techniques such as two-photon microscopy. Unlike these optical methods, however, ULM provides deeper tissue penetration, allowing access to brain regions that are otherwise difficult to visualize noninvasively.
ULM also offers functional imaging capabilities, enabling the quantitative assessment of blood flow velocity, perfusion dynamics, and vascular remodeling. These features make it highly suitable for investigating both physiological processes and pathological changes in neurovascular networks. Compared to MRI and PET, ULM is more cost-effective, does not require heavy infrastructure, and avoids the use of contrast agents with known toxicity risks, relying instead on gas-filled microbubbles with favorable safety profiles.
The combination of high spatial and temporal resolution makes ULM particularly valuable for a range of research applications. It is especially powerful in studies targeting microvascular structures and deep brain nuclei, where traditional modalities often fall short. Moreover, unlike histological approaches that require tissue fixation and staining, ULM enables real-time imaging in living animals, supporting longitudinal designs and dynamic interventions such as pharmacological challenges.
Beyond static vascular mapping, ULM also enables high-resolution functional imaging by capturing cerebral blood flow dynamics with exceptional spatial and temporal precision. Recent advances have demonstrated its feasibility for whole-brain functional neuroimaging in rodents, revealing stimulus-evoked and spontaneous activity-dependent changes in blood flow at the capillary level across distributed brain networks2. This positions ULM as a powerful tool for functional studies, effectively bridging the gap between mesoscale hemodynamic imaging and neuronal activity mapping.
Usability, efficiency, and reproducibility considerations
The ULM workflow described in this protocol has been optimized for ease of implementation and reproducibility across different experimental setups. The use of commercially available components, including FDA- and EMA-approved microbubbles and standard rodent anesthesia procedures, ensures wide accessibility. In addition, the integration of the entire processing pipeline within the dedicated software (IcoLab) greatly enhances usability by automating complex steps such as microbubble localization, trajectory reconstruction, and map generation. This streamlined interface reduces the need for custom code and minimizes user intervention, supporting consistent results across users and experiments. While high-resolution ULM processing is computationally intensive, especially for long acquisitions or volumetric data, batch-processing tools and GPU acceleration significantly reduce analysis time. The protocol's modular design also supports reproducibility, allowing researchers to fine-tune acquisition and processing parameters while maintaining a robust analytical framework. The protocol also includes a dedicated section on probe positioning using IcoScan and IcoStudio, which enables users to reliably reposition the probe at the exact same imaging slice across sessions, with an accuracy of approximately 100 µm. This feature is particularly valuable for longitudinal studies, where precise spatial consistency is essential for tracking vascular changes over time. These features collectively lower the barrier to entry for new users and support the scalable deployment of ULM in both exploratory and longitudinal imaging studies.
Conclusion
This protocol presents a complete workflow for implementing Ultrasound Localization Microscopy (ULM) in rodent brain imaging, covering surgical preparation, microbubble administration, image acquisition, and high-resolution vascular mapping using a standardized acquisition and analysis platform optimized for preclinical research. ULM represents a major advance in biomedical imaging, offering super-resolution in vivo visualization of the microvasculature with unprecedented detail. Compared to existing imaging modalities, ULM provides a unique combination of high spatial resolution, functional flow quantification, and accessibility, making it especially well-suited for studies of vascular development, neurovascular coupling, and disease pathophysiology. As the technology continues to mature, ULM is poised to become a cornerstone in both preclinical research and, eventually, clinical neuroimaging applications.

Though the brain accounts for only 2% of the body&#39;s mass, it consumes around 20% of its oxygen and glucose to sustain neuronal activity1. This high metabolic demand is met by an intricate vascular network, from large arteries and veins to microvessels like arterioles, venules, and capillaries, regulated through neurovascular coupling2. The disruption of this finely tuned process has been implicated in a broad spectrum of neurological disorders. Neurovascular uncoupling, for instance, is an early pathological feature in diseases such as Alzheimer&#39;s, Parkinson&#39;s, Huntington&#39;s, and Amyotrophic Lateral Sclerosis (ALS), often preceding the classical hallmarks of neurodegeneration such as amyloid-beta and tau pathology3,4,5,6. Imaging the brain&#39;s complex nature in vivo has therefore been of primary concern to address the growing need to understand physiological processes. Technological advancements have provided unique capabilities to probe brain function and activity, both directly and indirectly, with increasing precision, resolution, and accessibility.
Doppler ultrasound has emerged as a powerful tool for the functional measurement of brain activity using blood flow7. In particular, power Doppler imaging, designed to detect the motion of red blood cells (RBCs), transmits repeated ultrasonic pulses to blood vessels and records the amplitude of ultrasonic echoes backscattered by moving RBCs8. The advent of ultrafast ultrasound imaging, specifically functional ultrasound (fUS), marked a pivotal point in overcoming previous limitations. Indeed, conventional ultrasound scans tissue line by line-a sequential approach that confines its application to the major cerebral arteries, as the slow blood flows typical of the brain&#39;s microvasculature cannot be detected9. The more recent fUS technique uses multiple plane-wave transmissions to acquire thousands of frames per second, dramatically improving both the temporal resolution and signal-to-noise ratio (SNR). Crucially, fUS enables the detection of hemodynamic changes in very small vessels with velocities as low as 1 mm/s, supporting real-time functional imaging of brain activity down to the level of arterioles8.
While ultrafast Doppler methods have significantly improved microvascular imaging, they are still constrained by the diffraction-limited resolution of ultrasound10. In tandem with fUS, ultrasound localization microscopy (ULM) breaks through this impediment, enabling the visualization of microvascular structures at the capillary level by leveraging contrast agents and high-precision localization algorithms11. This provides unprecedented insights into vascular morphology and function. More specifically, in ULM, contrast agents made of stabilized gas-filled microbubbles, approximately 1-3 &#181;m in diameter, are injected into the bloodstream. They act as strong acoustic scatterers that can be detected as individual point sources. Given that microbubbles are significantly smaller than the ultrasound wavelength, they can be spatially and temporally isolated, appearing as the system&#39;s point-spread function (PSF). Inspired by optical super-resolution techniques such as STORM and PALM, sub-pixel localization of these microbubbles is achieved using deconvolution methods that map their positions with high precision10,12,13,14. This allows vascular structures to be resolved at a spatial resolution of 5-10 &#181;m, well beyond the diffraction-imposed limit of conventional ultrafast ultrasound power Doppler imaging.
ULM is a powerful and effective tool for vascular mapping down to the capillary scale and overcomes the limitations of many optical methods that are restricted to superficial layers. Its ability to perform microvascular imaging in living animals, allowing for longitudinal and pharmacological studies, is another key advantage of ULM over other techniques that require brain fixation and staining. The extracted information extends beyond static vascular maps, providing quantitative estimates of blood flow down to the capillary level, thereby opening promising avenues for both preclinical and clinical applications. To facilitate reproducibility, we describe a full workflow for ULM-based brain imaging in rodents, implemented using a standardized functional ultrasound imaging platform designed for preclinical research.

<table><tbody><tr><td>Animal temperature controller (heating plate coupled with a rectal probe)</td><td>Physitemp</td><td>TCAT-2DF</td><td></td></tr><tr><td>Atipamezole&amp;nbsp;</td><td>Orion Pharma, France</td><td>Antisedan</td><td>5 mg/mL injectable solution</td></tr><tr><td>C57BL/6 mice</td><td>Janvier Labs</td><td>SC-C57J-M</td><td>C57BL/6Rj male mice, 8 weeks old</td></tr><tr><td>Dental drill</td><td>Foredom, USA</td><td>K.1070 High Speed Rotary Micromotor Kit</td><td></td></tr><tr><td>Depilatory cream</td><td>Vichy</td><td>N/A</td><td></td></tr><tr><td>Eye ointment</td><td>TVM, UK</td><td>Ocry-gel</td><td></td></tr><tr><td>Hair trimmer</td><td>Phymep</td><td>ISIS GT 421 Aesculap</td><td></td></tr><tr><td>IcoLab processing software (v 1.2.0.0)</td><td>Iconeus, France</td><td>IcoLab</td><td></td></tr><tr><td>Iconeus One Xpand configuration</td><td>Iconeus, France</td><td>IconeusOne Xpand</td><td></td></tr><tr><td>IcoPrime 15MHz Probe</td><td>Iconeus, France</td><td>IcoPrime-15MHz Probe</td><td></td></tr><tr><td>IcoScan acquisition software (v1.9.0)</td><td>Iconeus, France</td><td>IcoScan</td><td></td></tr><tr><td>IcoStudio analysis software (v2.2.0)</td><td>Iconeus, France</td><td>IcoStudio</td><td></td></tr><tr><td>Iodine solution&amp;nbsp;</td><td>Mylan, France</td><td>N/A</td><td></td></tr><tr><td>Isoflurane anesthesia station</td><td>Minerve, Esternay, France</td><td>N/A</td><td></td></tr><tr><td>Ketamine</td><td>Virbac, France</td><td>Ketamine1000</td><td>100 mg/mL injectable solution</td></tr><tr><td>Meloxicam</td><td>Boehringer Ingelheim</td><td>Metacam</td><td>0.5 mg/mL injectable solution</td></tr><tr><td>Needles</td><td>Terumo, France</td><td>AN*1838R1</td><td>18 G x &amp;frac12;&amp;rdquo; needle with 8&amp;rdquo; tubing</td></tr><tr><td>Saline water</td><td>Laboratoire Gilbert Physiodose, Pharmacy, France</td><td>N/A</td><td></td></tr><tr><td>Scalpel</td><td>DREXCO medical, France</td><td>80011</td><td></td></tr><tr><td>SonoVue microbubbles</td><td>Bracco Imaging</td><td>SonoVue 8ul/ml</td><td></td></tr><tr><td>Spatula</td><td>Phymed, France.</td><td>K.1070 High Speed Rotary Micromotor Kit</td><td></td></tr><tr><td>Sprague-Dawley Rj:Han rats</td><td>Janvier Labs</td><td>RN-SD-M</td><td>RjHan:SD - Sprague Dawley male rats, 200-300 g</td></tr><tr><td>Stereotaxic frame</td><td>David Kopf (Tujunga, USA)</td><td>900-WA</td><td>Using Mouse Adaptor&amp;nbsp; (Ref: 922) and Non-Rupture Ear Bars (ref: 922)&amp;nbsp;</td></tr><tr><td>Syringe</td><td>DREXCO medical, France</td><td>2058</td><td></td></tr><tr><td>Ultrasound gel</td><td>DREXCO medical, France</td><td>Medi&#039;Gel</td><td></td></tr><tr><td>Winged infusion set</td><td>Terumo, France</td><td>SV*27EL</td><td></td></tr><tr><td>Xylazine 2%&amp;nbsp;</td><td>Bayer, France</td><td>Rompun</td><td>20 mg/mL injectable solution</td></tr></tbody></table>

ultrasound localization microscopy for super-resolution mapping of the rodent brain microvasculature

Ultrasound Localization Microscopy for Super-Resolution Mapping of the Rodent Brain Microvasculature

Ultrasound Localization Microscopy (ULM) is a super-resolution imaging technique that enables in vivo visualization of the brain&#39;s microvascular architecture surpassing&#160;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 &#181;m. 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&#160;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.

The representative examples in Figure 5 show a typical example of ULM data of a rat brain acquired over 1 min of recording following 200 µL of microbubble bolus injection, with a 10 µm resolution.
<img alt="Ultrasound imaging diagram: brain microbubble density, flow velocities, backscattered amplitude analysis." class="xfigimg" src="/files/ftp_upload/68612/68612fig5.jpg" data-altupdatedat="1778558422000" data-alt="Figure 5"> 
Figure 5: Representative ULM outputs acquired from a craniotomized rat following a single microbubble injection. Example maps of microbubble (MB) density (left), velocity (center), and backscattered amplitude (right) are shown for a full coronal brain slice, acquired over 1 min of recording following a 200 µL bolus injection of microbubbles. Scale bar: 2mm. Zoomed insets highlight the left cortical microvasculature (scale bar: 0.5 mm). The density map reflects the spatial distribution of MB occurrences, revealing the structure of the vascular network. The velocity map illustrates flow speed and directionality of individual MBs, enabling differentiation between vessel types. The amplitude map shows the backscattered ultrasound signal from MBs, providing an additional contrast mechanism that enhances visualization of vessel morphology and out-of-plane motion. <a href="https://www.jove.com/files/ftp_upload/68612/68612fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The resulting maps illustrate three complementary views of the cerebral microvasculature: microbubble (MB) density (left), velocity (center), and backscattered amplitude (right). Each map corresponds to a full coronal slice of the brain, with zoomed insets highlighting the left cortical microvasculature (scale bars: 2 mm and 0.5 mm, respectively).
The density map shows the spatial distribution of MB occurrences across the field of view. Vessel morphology is resolved far below the diffraction limit, revealing both large vessels and fine capillary structures. This type of map enables clear visualization of vascular network topology, including bifurcations and branching patterns, even in deep cortical regions.
The velocity map displays the flow speed of individual MBs projected along the axial (z) direction. This projection produces signed velocity values, where negative velocities reflect upward flow into the cortex and positive values reflect downward flow. In the rodent cerebral cortex, the vascular architecture is highly stereotyped: penetrating arterioles descend from the pial surface into the cortical layers, while venules ascend from the cortex back to the surface to drain deoxygenated blood16. This consistent anatomical arrangement enables the use of directional velocity measurements to distinguish between vessel types, with positive axial velocities typically indicating arterial flow and negative velocities indicating venous drainage. Overall, the velocity values measured with ULM are consistent with previously reported ranges in the literature10,17, spanning from 10-20 mm/s in penetrating arterioles to several centimeters per second in major intracranial arteries. These findings are in line with earlier observations that flowing microbubbles closely mimic the rheological behavior of red blood cells18, validating the use of ULM for quantifying physiological blood flow dynamics at the microscale.
The backscattered amplitude (BSA) map provides a spatial representation of the ultrasound signal intensity returned by each MB. As backscattered energy is maximized when MBs pass through the focal region, this map offers contrast based on elevational position. It helps refine segmentation by identifying out-of-plane motion or low-amplitude vessels, particularly at greater depths or near the focal edges. BSA maps are also valuable for detecting depth-related signal attenuation or identifying mismatches in focal alignment15.
To demonstrate the reproducibility of plane targeting with the Brain Positioning System, Figure 6A shows ULM vascular density maps acquired in the same mouse under anesthesia on day 0 (Week 1) and day 7 (Week 2), targeting the same coronal plane. The overlay of both maps shows a strong spatial correspondence of the vascular signal, confirming the precision of plane repositioning and the reliability of longitudinal experiments, as previously shown19.
Quantitative analysis can be applied to any of these outputs. For instance, users may segment vessels using intensity thresholds or clustering algorithms, and then extract regional statistics such as vessel density, average flow speed, and morphometric parameters including vessel radius, average length, number of branches, and other structural descriptors of the vascular network. Comparisons across cortical regions or experimental groups (e.g., drug-injected vs. control) can be performed directly on these maps or through derived parametric statistics on commonly identified vessels20,21. As an example of such analysis, we performed regional quantifications of vascular parameters in the left somatosensory cortex (S1BF, Figure 6B) and left thalamus (Figure 6C). The density maps were first binarized, skeletonized, and vessel radii were estimated using a Euclidean distance transform. Flow velocities were directly obtained from the MB trajectories, and flow rate was derived from the vessel radius using Poiseuille's law as previously detailed21. The same methodology as described in that study was applied here. Whisker plots display the distributions of vessel radius, velocity, and flow rate for d0 and d7, demonstrating good reproducibility across sessions. Together, these multimodal ULM outputs enable comprehensive characterization of the brain's microvascular network, offering both anatomical and dynamic insights with unprecedented resolution.
<img alt="Brain vasculature analysis; imaging data, box plots; radius, velocity, flow rate comparisons." class="xfigimg" src="/files/ftp_upload/68612/68612fig6.jpg" data-altupdatedat="1778558422000" data-alt="Figure 6"> 
Figure 6: Longitudinal reproducibility of plane targeting and example of vascular quantifications using the Brain Positioning System. (A) ULM vascular density maps acquired from the same anesthetized mouse on day 0 (Week 1) and day 7 (Week 2), targeting the same coronal plane using the Brain Positioning System (scale bar = 1 cm). The overlay of both maps shows a strong spatial correspondence of the vascular signal, illustrating the reproducibility of plane positioning across sessions. (B,C) Representative quantification of vascular parameters extracted from two regions of interest: left somatosensory cortex (S1BF, B) and left thalamus (C). For each region, we display the vascular density map at d0 (scale bar = 0.5 cm), together with whisker plots showing the distribution of vessel radius (µm), flow velocity (mm/s), and flow rate (mm3/s) at d0 and d7 (median, 25th-75th percentile, whiskers = 1.5×IQR, outliers plotted individually). These results illustrate the ability of the pipeline to extract morphometric and hemodynamic metrics from the ULM data. <a href="https://www.jove.com/files/ftp_upload/68612/68612fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Ultrasound Localization Microscopy (ULM) is a recently developed technique that allows for an unprecedented level of resolution in imaging the microvasculature in vivo. In this work, we describe in detail how to obtain super-resolved images of the brain microvasculature in rodents using a standardized functional ultrasound imaging platform designed for preclinical research.

Ultrasound Localization Microscopy (ULM) is a super-resolution imaging technique that enables in vivo visualization of the brain&#39;s microvascular ...

Our research aims to characterize the brain microvascular architecture and flow, while refining super-resolution ultrasound methods. Recent developments have optimized microbubbles tracking, and strengthened processing pipelines to create more stable protocols for super-resolution vascular mapping with ULM. To begin, place an anesthetized mouse onto a stereotaxic frame with a heating pad set to 37 degrees Celsius.Using firm but non-damaging pressure, pinch the toe or tail to check for the absence of reflexes and confirm adequate anesthesia depth. Apply eye ointment to both eyes to prevent eye ulceration and cataract formation during the procedure. Now, shave the head of the mouse using a trimmer.Apply depilatory cream. Let it sit for one to 1.5 minutes maximum, then rinse thoroughly with warm water, and dry with gauze. Next, apply centrifuged ultrasound gel onto the head to ensure acoustic coupling.For tail vein catheterization, use a 27 gauge half-inch winged infusion set connected to an 18 gauge half-inch needle with eight inches of tubing. Shorten the tubing, if possible, to minimize dead volume. Gently turn the animal's tail 90 degrees to expose the lateral tail veins.Then secure the tail in place with tape to maintain stability and prevent movement during catheterization. Now, identify the tail vein. To improve vein visibility, warm the tail with lukewarm water to promote vasodilation.Using a finger, apply gentle pressure at the base of the tail, and slide it toward the injection site against the direction of blood flow. Insert the needle parallel to the vein with the bevel facing up. Observe the tubing for blood flashback to confirm correct needle placement.On the imaging system, launch the functional ultrasound acquisition software and create a new experiment session. Adjust the position of the ultrasound probe. Now, start the live view acquisition in the software, and adjust the probe in real time using Power Doppler imaging to center the brain on the screen.Optimize the imaging parameters, such as contrast and compression, to enhance visualization quality. Then, open the Angio 3D menu in the acquisition software. Adjust the scanning parameters to fully cover the brain, then start the scan acquisition.Keep the acquisition software open, and once the Angio 3D is done, launch the IcoStudio software for analysis and visualization. Load the Angio 3D scan for review. Navigate to the brain registration panel, and register the Angio 3D scan using either the fully automatic or manual mode.Save the completed registration as a BPS file. Now, open the Brain navigation menu, and scroll down to the Atlas manager panel. Use the parent and child tree navigator to browse the Allen Mouse brain atlas, and select the desired anatomical regions for overlay in the three-view panel.Choose an imaging plane that overlaps with the selected regions of interest. In the coronal view, manually place two markers to define the desired imaging slice. Then, click on BPS Positioning to extract the motor coordinates for the selected imaging plane.Review the image preview from the Angio 3D scan, and click Copy BPS coordinates. Switch to the IcoScan software, and open the Move probe panel. Click on Neuronavigation, paste the copied values, and allow the system to automatically align the probe with the selected imaging plane.Then, start live imaging to confirm that the current imaging plane aligns with the previously selected plane. Using a sterile syringe, inject five milliliters of 0.9%sodium chloride solution into the ultrasound contrast agent microbubble vial through the septum. Agitate the vial vigorously for 20 seconds to resuspend the microbubbles and ensure uniform dispersion.Predefine the recording parameters, including ultrasound sequence, frame rate, and total recording duration. Then, draw the desired volume of microbubbles for injection and begin the acquisition. Once acquisition starts, inject 100 microliters for mice using the appropriate dilution.Confirm contrast enhancement on the live visualization panel. Immediately flush the remaining microbubbles in the dead volume by injecting physiological fluid into the catheter. Wait for the acquisition to finish, and save the recorded data.Open the Icolab software, and click on the Ultrasound localization microscopy tab. Then, click on Compute ULM maps to begin the processing workflow. Select the source folder that contains the previously acquired data.Then, select the specific scan file to process and adjust the processing time boundaries as needed. Click on Next to move to the reporting step. Set the output folder path for saving the processed files, and configure the visualization parameters for the report images.Now click on Run to begin processing the data. Once completed, a TRK file will be created containing localized microbubble coordinates, along with TIFF files representing density, velocity, and backscattered amplitude. Load the TRK file into IcoStudio to begin exploring the processed data.Then, use the right side panel in IcoStudio to adjust visualization parameters such as contrast, compression, and color map for enhanced data representation. To set the integration time, adjust the double slider located at the bottom of the main view to define the start and end frames for the analysis. The density tab shows the distribution of microbubble occurrences.Choose between absolute density and directional density. After that, click on the Velocity tab to display microbubble speed in millimeters per second using absolute, directional, axial and lateral modes. Adjust the contrast, compression and velocity thresholds to improve image clarity.Then explore the Backscattered Amplitude tab to view microbubble reflectivity, which enhances contrast, and reveals microvascular structures and out-of-plane motion. Export images as high-resolution PNGs, or generate TIFF files for quantitative analysis with customizable pixel size. Microbubble density maps of a coronal rat brain slice acquired with ULM resolved both large vessels and fine capillary structures, revealing detailed vascular topology, including bifurcations and branching patterns.The velocity map of the same brain slice showed clear axial directional flow, with upward flow representing venous drainage, and downward flow corresponding to arterial perfusion, consistent with known cortical vascular architecture. The backscattered amplitude map provided depth-dependent contrast, and highlighted vessels based on their elevational position, useful for segmenting out-of-plane or low-amplitude vessels. Vascular density maps acquired one weak apart in the same mouse showed strong spatial overlap, confirming the reproducibility of coronal plane targeting using the brain positioning system.Quantitative analysis of the left somatosensory cortex across sessions showed similar distributions in vessel radius, velocity and flow rate, demonstrating measurement reproducibility. Similar reproducibility was observed in the left thalamus region, with consistent values for vessel radius, velocity, and flow rate between week-one and week-two sessions. This work establishes that ultrasound localization microscopy can reliably produce super-resolution maps of microvascular structure and flow in vivo.This protocol fills the need for a standardized procedure that enables robust ULM acquisitions in living rodents using a dedicated functional ultrasound system.

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Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

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