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

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.