Pipeline for Planning and Execution of Transcranial Ultrasound Neuromodulation Experiments in Humans

Summary

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique that requires careful planning of acoustic and thermal simulations. The methodology describes an image processing and ultrasound simulation pipeline for efficient, user-friendly, streamlined planning for human TUS experimentation.

Abstract

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique capable of manipulating both cortical and subcortical structures with high precision. Conducting experiments involving humans necessitates careful planning of acoustic and thermal simulations. This planning is essential to adjust for bone interference with the ultrasound beam's shape and trajectory and to ensure TUS parameters meet safety requirements. T1- and T2-weighted, along with zero-time echo (ZTE) magnetic resonance imaging (MRI) scans with 1 mm isotropic resolution, are acquired (alternatively computed tomography x-ray (CT) scans) for skull reconstruction and simulations. Target and trajectory mapping are performed using a neuronavigational platform. SimNIBS is used for the initial segmentation of the skull, skin, and brain tissues. Simulation of TUS is carried over with the BabelBrain tool, which uses the ZTE scan to produce synthetic CT images of the skull to be converted into acoustic properties. We use a phased array ultrasound transducer with electrical steering capabilities. Z-steering is adjusted to ensure that the target depth is reached. Other transducer configurations are also supported in the planning tool. Thermal simulations are run to ensure temperature and mechanical index requirements are within the acoustic guidelines for TUS in human subjects as recommended by the FDA. During TUS delivery sessions, a mechanical arm assists in the movement of the transducer to the required location using a frameless stereotactic localization system.

Introduction

Commonly used non-invasive neurostimulation techniques include transcranial direct current stimulation (tDCS) and transcranial magnetic stimulation (TMS). However, both have limited penetration depth and low precision^1,2. By contrast, transcranial ultrasound (TUS) is an emerging non-invasive technique capable of enhancing or suppressing neuronal activity^3,4,5 and targeting cortical or subcortical structures at millimeter precision^6,7. Animal models using rodents^4,8,9, rabbits¹⁰, sheep^5,11, swine⁶, and nonhuman primates^7,12,13,14 have shown the efficacy and safety of TUS. Studies have demonstrated that targeting various brain regions can elicit limb movements⁸ in rats, somatosensory evoked potentials (SSEPs) in swine⁶, and changes in visuomotor activity¹², cognitive, and motivational decision-making in nonhuman primates¹³ among other changes in behavior. In humans, TUS has been observed to change motor evoked potentials (MEPs) and performance on a reaction time task when targeting the primary motor cortex^15,16 and altered performance on a tactile discrimination task and SSEPs when targeting the somatosensory cortex¹⁷ and sensory thalamus¹⁸. Histological analyses have revealed no gross or microscopic structural changes associated with TUS in swine⁶, sheep^5,11, rabbits¹⁰, and nonhuman primates¹⁴, and no side effects have been seen that significantly differ from other non-invasive neurostimulation techniques¹⁹.

TUS uses pulsed low-intensity focused ultrasound at a frequency between 200 kHz and 700 kHz to produce a transient neuromodulatory effect. The typical spatial-peak pulse-average intensity (I_sppa) in situ is 10 W/cm² or less, with reported duty cycles (percentage of time when ultrasound is on) ranging from 0.5% to 70% in humans^{20,21,22,23,24}. Although the mechanisms of TUS neuromodulation have been proposed to mainly involve mechanical agitation of lipid membranes leading to the opening of ion channels^25,26,27, possible thermal and cavitation effects cannot be ignored. They are assessed through mechanical (MI) and thermal (TI) indices. The MI describes the predicted cavitation-related bioeffects that will occur with TUS, whereas the TI describes the potential temperature increase within tissues following ultrasound application^28,29. Furthermore, changing the frequency and input intensity also causes the MI and TI to change. Higher frequencies have better spatial resolution and decrease the probability of mechanical bio-effects; however, they have stronger absorption in the tissue, which increases the potential for temperature rise²⁸. Alternatively, lower frequencies at the same intensity increase the MI. Similarly, increasing the intensity tends to increase the magnitude of mechanical and thermal bio-effects³⁰. It is, therefore, imperative that careful planning and simulation be performed before experimentation sessions for all TUS parameters that will be implemented.

Planning a TUS experiment requires the identification of the target and trajectory of interest and the performance of thermal and acoustic simulations. Simulations assist in optimizing mechanical effects and mitigating the thermal effects of TUS on tissues. They require understanding the prediction of skull heating, pressure amplitude of the ultrasound at the focal point, focal correction, and other heating within the skull and skin. Adequate simulation ensures the focal point will reach the target of interest and safety parameters for ultrasound use set out by the safety guidelines on biophysical safety as recommended by the International Transcranial Ultrasonic Stimulation Safety and Standards Consortium (ITRUSST)³¹, which are based on FDA and Health Canada recommendations, are followed. Recent studies have also highlighted an auditory confounding effect accompanied by TUS^32,33,34 in animals and humans, whereby TUS stimulation can activate auditory pathways in the brain to elicit responses^32,33,34. Transection of the auditory nerves³², removal of cochlear fluid³², or chemical deafness³³ in rodents have been employed to diminish these effects in animals. In humans, administering an auditory tone through headphones has been used to effectively mask auditory noise from TUS, controlling for the TUS-induced auditory activity confound³⁴. This highlights the need to control for auditory noise in sham stimulation conditions, which must be incorporated into protocol planning, design, and implementation.

Here, we present a guide on how to appropriately complete the preparation (step 1, step 2), planning (step 3), simulations (step 4), and TUS delivery (step 5) required to perform TUS neuromodulation experiment in humans.

Protocol

All methods involving the use of human subjects were performed in compliance with the Tri-Council Ethical Conduct for Research Involving Humans, and the protocol was approved by the Conjoint Health Research Ethics Board (CHREB) at the University of Calgary. All subjects provided informed written consent before participation. Human participants were required to be healthy, right-handed adults between ages 18 and 40 willing and able to complete a magnetic resonance imaging (MRI) scan. Exclusion criteria included family history of seizure, mood, or cardiovascular disorders, ear trauma, alcohol or drug dependency, use of prescription medications, metal implants including a pacemaker, pregnancy, cardiovascular disorders, history of neurological or psychiatric disorder, inability to communicate with investigator and study staff, and legal incapacity or limited legal capacity. The protocol described below follows the recommendations by ITRUSST on the standardized reporting of TUS studies³⁵. The details of the equipment, software, and necessary weblinks used in this study are listed in the Table of Materials.

1. High-resolution magnetic resonance imaging

1. Image the participant using a 24-channel head/neck coil.
   NOTE: Alternatively, the best head/neck coil available at the site is sufficient to obtain an anatomical MRI.
2. Collect T1- and T2-weighted MR images, and Zero-time echo (ZTE) sequences at 1 mm resolution.
   NOTE: It is recommended that MR images are collected without the participant wearing over-ear headphones, as it can impact image reconstruction and make subsequent planning and implementation steps difficult. Instead, it is recommended that the participant only wear earplugs for MR safety.
   1. For T1-weighted imaging, use a 3D inversion recovery-prepared fast spoiled gradient echo (FSPGR) sequence with the following parameters: repetition time (TR) = 8.4 ms, echo time (TE) = 3.2 ms, inversion time (TI) = 650 ms, flip angle of 10 degrees, a field of view measuring 256 mm x 256 mm x 188 mm, matrix size of 256 x 256 x 188, GRAPPA (ARC) factor of 2 in the phase encode direction, and sagittal-oblique orientation to approximate alignment with AC-PC.
      NOTE: The total scan time is 5 min and 21 s.
   2. For T2-weighted imaging, use a 3D fast-spin echo (FSE) with the following parameters: TR = 3000 ms, TE = 60-90 ms, an echo train length of 130, the field of view measuring 256 mm x 225 mm x 188 mm, matrix size of 256 x 256 x 188, and a sagittal-oblique orientation approximating alignment with the AC-PC axis. A GRAPPA (ARC) factor of two was utilized in the phase encode and slice direction.
      NOTE: The total scan time is 3 min and 58 s.
   3. For ZTE images, use an isotropic 3D ZTE scan with the following settings: TR = 698 ms, TE = 16 µs, flip angle of 1 degree, receiver bandwidth of 62.5 kHz, number of averages = 3.5, a field of view measuring 256 mm x 256 mm x 256 mm, matrix size of 256 x 256 x 256.
      NOTE: The total scan time is 5 min and 23 s.
      NOTE: MR images must be completed before TUS planning can commence.

2. Pre-processing participant images

1. Convert T1-w, T2-w, and ZTE DICOM scan files from DICOM to Nifti format using the dcm2niix tool. In a terminal window, execute: dcm2niix <Path to DICOM scan files>.
   NOTE: The dcm2niix tool is available on GitHub (see Table of Materials). The BabelBrain tool co-registers the participant's images to accommodate for images collected on separate days. If T1-w images are not isotropic, they are resampled to 1 mm isotropic voxels; however, CT/ZTE images are not. Instead, the masks for the simulation are resampled to the final resolution in terms of PPW. No additional co-registration or alignment steps are required.
2. Execute co-registration and tissue mask extraction using SimNIBS's charm tool. In a terminal window, execute: charm <ID> <Path to T1-W Nifti file> <Path to T2-W Nifti file> --forceqform, where <ID> is a string for identification. At this point, the experiment planning can be paused and continued at another time.
   NOTE: The files created will be saved in an m2m <ID> subdirectory, which will be used in subsequent planning stages. Pre-processing images with the charm tool is necessary for tissue segmentation purposes, as this is required for use in the BabelBrain tool³⁶.

3. Trajectory planning

1. Open Brainsight, click on New Empty Project, and load the participant's T1-w Nifti image, produced in step 2.1.
   NOTE: If using a Brainsight version before v2.5.3, either "New Empty Project" or "New SimNIBS Project" can be chosen. If using Brainsight v2.5.3 onwards, there is integration between Brainsight and BabelBrain and it is highly recommended to click "New SimNIBS Project"; however, both are still sufficient to complete planning steps. "New Empty Project" requires loading the T1-w image, while "New SimNIBS Project" requires loading a .msh file that directly loads the T1-w image. All other functionalities and steps remain the same.
2. Click on Overlays, and then on Configure Overlays to create and view an overlay. Click on Add to select the file to overlay.
   NOTE: If the target cannot be visualized on a T1-w image, it must first be localized on a co-registered T2-w image (so the T1-w and T2-w coordinates are the same). Then, transfer the coordinates onto a T1-w image for subsequent planning steps. The charm tool in step 2.2 produces a co-registered T2-w image in the m2m <ID>/T2_reg.nii.gz file path.
3. Close the overlays section, click on the Targets tab, followed by Configure Targets. Click on the blue information button to change the opacity, colour, and threshold of the overlay to allow for a comparison of the images.
   NOTE: This can be performed to visualize co-registered Nifti images and should be used to observe how the acoustic simulations will overlay with the target.
4. Click on the Reconstructions tab, then on the New Reconstruction… drop-down menu. Click on Skin, followed by Compute Skin in the new window. Once complete, click on the close button in the top left corner of the window.
5. Within the "Reconstructions" tab, click on the New Reconstruction… drop-down menu followed by Full Brain Curvilinear.
   1. Adjust each box on the sagittal (Figure 1A), coronal (Figure 1B), and transverse (Figure 1C) images by dragging the edges of the green box, so the lines are tightly surrounding the brain (Figure 1).
   2. Scroll through all the slices to ensure no edges of the tissue are overlapping. Click on Compute Curvilinear and adjust the peel depth to 4 mm (Figure 1D).
      NOTE: The full brain curvilinear provides a 3D reconstruction of the cortex, which can be peeled to observe different depths. It allows for accurate and consistent placement of a TMS coil over the brain to localize the region of interest.
6. Click on the Landmarks tab on Brainsight, followed by Configure Landmarks.
   1. Place the crosshairs (cursor) on the tip of the nose, and in the "Name:" field label the landmark "Nose" (Figure 2A). Place the crosshairs between the eyes above the bridge of the nose and label the landmark "Nasion" (Figure 2A).
   2. Place the crosshairs on the left ear and label the landmark "L Ear" (Figure 2B). Place the crosshairs on the right ear and label the landmark "R Ear" (Figure 2C).
7. Click on the Targets tab and use the crosshairs (cursor) to localize the target of interest.
   1. Place the crosshairs (cursor) on the location of the desired target and use the angle toggles on the right side of the screen to set the trajectory angle.
   2. Once the desired target and trajectory are selected, click on the New drop-down menu, and choose Trajectory. Name the target by typing into the text box next to "Name:".
   3. Export the target by clicking on Export… and save it in the appropriate subject folder. Once the file is saved, the planning can pause, and the experiment can restart later.
      NOTE: The target will be used in subsequent steps to complete thermal and acoustic simulations.

Figure 1: Creating a full brain curvilinear in Brainsight. (A) Box adjusted to the edge of the sagittal MR image. (B) Box adjusted to the edge of the coronal MR image. (C) Box adjusted to the edge of the transverse MR image. (D) Full brain curvilinear reconstruction with a peel depth of 4 mm. Please click here to view a larger version of this figure.

Figure 2: Landmarks placed on the skin reconstruction and MR image. (A) Nose and nasion landmarks placement. (B) Left ear landmark placement. (C) Right ear landmark placement. Please click here to view a larger version of this figure.

4. Simulations with BabelBrain

NOTE: Details for simulation with BabelBrain can be consulted in BabelBrain's manual: https://proteusmrighifu.github.io/BabelBrain/index.html.

1. Open BabelBrain and select the required files for acoustic and thermal simulations.
   NOTE: This submission uses a phased array transducer; however, other transducers are fully compatible with this planning software. This program can complete simulations for Single, CTX_500, H-317, H-246, and transducers. In the following method, the H-317 will be used as the selected transducer.
   1. Select the txt file previously exported from Brainsight during step 3.7.3 by clicking on Select Trajectory… and choosing the appropriate participant file. Click on Select SimNIBS… to choose the SimNIBS file (m2m_folder name) created in Step 2. Click on Select T1W… and choose the T1-w image produced in step 2.1 used previously for target and trajectory mapping.
   2. Within the drop-down beside the "Use CT?" label, choose real CT if using a CT scan, ZTE if using a ZTE scan, or NO if using a simplified mask generated by the charm tool if only T1- and T2-w images are available. When using a CT or ZTE image, click on the Correg.? drop-down menu followed by CT to MR. Click on Select and choose the corresponding image from the appropriate participant file.
   3. Click "Select Thermal Profile …" and choose the thermal profile file that describes the experiment's sonication parameters in terms of duration on, duration off, and duty cycle.
      NOTE: Details can be consulted in BabelBrain's manual: https://proteusmrighifu.github.io/BabelBrain/index.html. Examples of profiles are available at https://github.com/ProteusMRIgHIFU/BabelBrain/tree/main/Profiles.
   4. Click on the drop-down menu beside "Transducer" and select the transducer used for experimentation. Click on the drop-down menu beside "Computing backend" and choose the computing backend of the computer running the simulations. Once all information is entered, click on CONTINUE to complete the rest of the simulations.
2. Sub-step A: Calculate Mask.
   1. Select the ultrasound frequency of the transducer and the appropriate point per wavelength (PPW).
      NOTE: 6 PPW is enough for most scenarios.
   2. Leave the normalized ZTE range and Hounsfield units (HU) as are.
      NOTE: If using a real CT, only the HU threshold will appear. If no ZTE or CT scan is provided, this entry will be absent.
   3. Click on Calculate planning mask. Inspect the image, checking to ensure the bounds for the skin, skull, and brain were accurately recognized.
      NOTE: If any discrepancies are present, adjust the normalized ZTE range and/or HU threshold.
3. Sub-step B: Acoustic Simulation. Click on the Step 2 - Ac Sim tab to open.
   NOTE: This tab will look different depending on the selected transducer. What follows is specific to the H-317 transducer.
   1. Adjust the distance from the cone to focus to reflect the distance from the cone's surface to the target by typing in the appropriate distance.
      NOTE: This is dictated by the physical cone to be used in experiments.
   2. Execute the simulation by clicking on the Calculate Fields button.
   3. Adjust the Z steering (mm) value so the crosshairs (indicating the target) are in the center of the focal point (Figure 3). Press the up or down arrows or manually enter the required value, then click on Calculate Fields.
      NOTE: Positive values will direct the focal spot deeper into the brain, whereas negative values will cause the focal spot to move superficially.
   4. Adjust X/Y mechanical if the focal spot is lateral to the intended target. Leave Z mechanical as is. Click on Calculate Fields.
      NOTE: Z mechanical is recalculated as a function of the distance from the cone in 4.3.1. If required to adjust the Z steering, X/Y mechanical, or maximal depth beyond the target after completing a calculation, acoustic simulation files must be re-calculated. Click on Calculate Fields and select Yes to re-calculate. Select No to re-load the existing files.
4. Sub-step C: Thermal Simulation. Click on the Step 3 - Thermal Sim tab, then click on Calculate Thermal Fields. Assess the mechanical and thermal simulations (Figure 4) to ensure they meet the acoustic guidelines in human subjects as recommended by ITRUSST³¹ (Mechanical index (MI) in soft tissue ≤1.9; thermal rise ≤2 °C, thermal dose ≤0.25 CEM43, exposure time restricted to 80 min for 1.5 < cranial thermal index (TIC) ≤ 2.0, 40 min for 2.0 < TIC ≤ 2.5, 10 min for 2.5 < TIC ≤ 3.0, 160 s for 3.0 < TIC ≤ 4.0, 40 s for 4.0 < TIC ≤ 5.0, and 10 s for 5.0 < TIC ≤ 6.0).
   NOTE: BabelBrain solves the Bioheat Transfer Equation to estimate the rise of temperature³⁶. In rare occasions, abnormal high-temperature voxels may occur, in which case MR images should be analyzed if any anatomical features could explain the unusual temperature rise.
   1. Adjust the I_sppa by clicking on the up or down arrows beside the Isppa(W/cm²) box or manually entering a value above 0.1 to observe how the parameters change with different intensities.
      NOTE: A summary of all intensities, temperature changes, and mechanical indices can be exported as a CSV file for reference. The software calculates the required intensity in water conditions (I_sppa in water) that needs to be applied to achieve the desired intensity in situ.
   2. Click on the MTB, MTS, and MTC buttons in the lower region of the interface to view the slice with the highest temperature in the brain, skin, and skull. After the thermal simulation step is finished, simulations are complete.
      NOTE: Files will be saved automatically in the same location as the T1-w image.
      ​NOTE: Acoustic files with an ending <…FullElasticSolution_Sub_NORM.nii.gz> can be loaded in Brainsight to visualize the acoustic field overlay in T1 space. There will be files for water-only conditions (with the infix "Water" in the filename) and files for tissue-present conditions (no infix in the filename). Follow the steps in the note following step 3.1 to add an overlay. After the thermal simulation step, simulations are complete, and the experiment can be paused until the TUS delivery session.

Figure 3: Acoustic simulation using BabelBrain. Please click here to view a larger version of this figure.

Figure 4: Thermal simulation using BabelBrain. Please click here to view a larger version of this figure.

5. TUS delivery session

1. Open Brainsight and click on Open Existing Project. Select the Brainsight file created and saved during trajectory and target mapping.
2. Click on Sessions to begin a new experimentation session. Click on the drop- down and select New, followed by Online Session. Click on the target name followed by Add, and Next to add the target to the experiment session and continue to the experimentation window.
   NOTE: If TUS delivery is performed using a different computer than what was used for the planning, Brainsight will require the appropriate T1-w image to be chosen. In this case, a new window will open and prompt you to select the associated T1-w participant image from their participant file.
3. Click on Window, and Tool Calibration, and select the tools to be used, followed by Re-Calibrate.
   NOTE: This step must be completed for the transducer and all other tools used during the experimental session, such as a TMS coil.
   1. Secure the large coil tracker and the calibration block to the transducer. Ensure the camera can see the fiducials on both tools. Click on Begin Calibration Countdown to re-calibrate.
      NOTE: Saving the calibration will ensure it remains the same for that day of experiments. If this is not performed and the computer turns off, the equipment will require re-calibration.
4. Set up the transducer according to its set-up procedures for experimentation. Remove the tracker on the transducer before setting it up so it is not damaged.
   NOTE: The set-up described is for the H-317 transducer with a customized coupling cone.
   1. Assemble the cone as required, placing the transducer film between the cone and the end cap. Attach the coupling cone to the flange on the transducer using screws, washers, and nuts, ensuring all are tight so there are no leaks.
   2. Fill the transducer with de-ionized water and attach the tubes from the pump to the transducer, connecting the tube labeled "IN" to "OUT" and the "OUT" to "IN". Plug the water hole while pressing "DRAIN" on the pump to relieve pressure.
   3. Flip the transducer upside down to isolate the air bubbles on the film, then rotate upwards, allowing them to exit through the transducer tube labeled "OUT". Repeat this until all visible air bubbles are removed. Set up the transducer so its "OUT" tube is angled forward and at the highest point, and leave with the pump running for 30 min.
   4. Flip the transducer upside down to verify that all air bubbles have been removed. If some remain, isolate the air bubbles on the film, and rotate the transducer as before to ensure they exit through the "OUT" tube. Repeat until all bubbles have been removed.
   5. Turn off the pump, detach the hoses, and lock the transducer at neutral. Re-attach the tracker to the transducer and proceed with participant set-up.
5. Attach the subject tracker to the participant's head using either glasses, a headband placed above the ears and eyebrows (Figure 5), or the lightweight adhesive tracker. Ensure the fiducials are oriented so they are visible to the camera and will not be blocked or hit by the transducer when moving towards or once it has reached its final position.
   1. Place the camera in front of the participant so it can see the subject tracker and the pointer.
      NOTE: If they are not visible, the indicator at the bottom left of the screen will be red. This can also be verified by entering the "Polaris" tab on Brainsight.
6. Click on the Registration tab in Brainsight. Place the pointer on all four landmarks set during step 3.6, stabilizing it with both hands, then click on Sample & Go To Next Landmark.
   NOTE: It is important that the headband or glasses remain tight on the participant's head and that the subject tracker does not move. If this happens, perform the registration again. If the fiducials must be rotated to accommodate transducer movement after registration has been completed, re-do the registration.
7. Click on the Validation tab to validate the participant registration. Lightly place the pointer on various positions along the scalp and ensure all points are less than 3 mm.
   NOTE: Commercial neuronavigation systems created for TMS are being utilized for TUS experimentation in humans. A 3 mm translational error is acceptable for handheld transducer calibration³⁷, as it is consistent with target accuracy^38,39 and other neuronavigation systems⁴⁰. The 3 mm value is embedded into the Brainsight neuronavigation software.
   1. If any points are greater than 3 mm, click on the Add button to add additional landmark points for up to a maximum of three points. If more than three points are required to achieve a consistent validation under 3 mm, re-do the registration.
      NOTE: To improve the success of the registration, it is recommended that MR images are collected without the participant wearing over-ear headphones, as it can impact image reconstruction and make registration difficult. Be cautious that the pointer is placed as close to the landmark as possible before clicking on Sample & Go To Next Landmark, and ensure that the pointer is stabilized using both hands. The validation provides reassurance that the average of all points along the scalp surface is below the threshold. Do not proceed until all random locations on the scalp are below the 3 mm threshold.
8. Secure the participant's head with the chin rest and stabilizer placed on the back of the head to prevent movement (Figure 5). Ensure they are seated comfortably in the chair for the duration of the experiment.
9. Click on Perform. Use the mechanical arm to position the transducer over the target of interest along the selected trajectory.
   NOTE: Ensure to practice and be familiar with how the components of the mechanical arm move to reach the target. Success in positioning the mechanical arm comes through understanding the mobility of the arm and being familiar with how it moves about the head orientation, as this is highly dependent on the location of the target.
   1. Use the crosshairs on the bullseye window to achieve the correct positioning and angle (Figure 6). Align both circles (translational and rotational) so they are positioned on the crosshairs and refer to the accuracy reading for verification.
   2. Part the subject's hair along its natural part and comb in the ultrasound gel to ensure there are no air pockets within the hair. If a large area requires coupling, apply ultrasound gel to the transducer film. Fill any remaining air pockets with additional ultrasound gel using a syringe.
   3. Lower the transducer to the appropriate height by observing when the crosshairs are in the middle of the box surrounding the target (Figure 6). This will indicate when the appropriate target depth has been reached.
10. A custom-made Python script uses the results from simulations in step 4 to program the 128-amplifier unit and generate sound through headphones (https://github.com/ProteusMRIgHIFU/TUSApp). If performing an online and/or double-blind experiment, ensure that masking audio is played through headphones^34,41 or ramp the stimulus on- and offset over the burst duration^42,43,44 as methods to reduce potential auditory confounding effects.
    NOTE: At this step, if using a commercially available unit, such as a CTX-500 transducer, prescribe the intensity that was simulated in water conditions, along with other parameters (PRF, duration, duty cycle). The H-317 transducer uses a dedicated script to run the transducer; therefore, that step is omitted from this submission.
    1. Generate the masking audio in MATLAB to play upon the start of TUS delivery. Sound during sham stimulation combines a continuous sin wave at 250 kHz, a square wave at a burst frequency of 100 Hz sampled at 48 kHz, with random noise to closely replicate the sound produced from the transducer. During TUS, only random noise plays through the headphones, as the sound emitted from the transducer will also be heard by the subject.
11. Following completion of TUS delivery, remove the transducer, gel, and tracker from the participant's head before any subsequent experimental procedures.
12. To clean up the TUS equipment, remove the tracker from the transducer to avoid damage. Disassemble the transducer by removing the plug on the top and flipping it upside down to drain the water. Remove the coupling cone and disassemble it to allow the pieces to air dry.
    1. Return the transducer to neutral and ensure all arm components are locked tight at 90˚ for storage until the next TUS experimental session.

Figure 5: Fiducials to be used for neuronavigation. Glasses (left) and headband (right) with fiducials attached for subject tracking. The subject's head is secured with a chin rest and stabilizer behind their head. Please click here to view a larger version of this figure.

Figure 6: Brainsight neuronavigation screen for target localization during TUS delivery session for a primary motor cortex (M1) target. Rotational and translational indicators guide the experimenter on where to position the transducer over the scalp and when the trajectory angle has been achieved. Accuracy to target indicates how close the translational and rotational orientation is to the target and should be used to fine-tune movement. The depth indicator shows the depth of the focal spot and should be used to lower the transducer to the appropriate height. Please click here to view a larger version of this figure.

Results

Figure 7 illustrates comparative session samples from one of our studies⁴², featuring two distinct participants employing specific ultrasound parameters (fundamental frequency of 250 kHz, sonication duration of 120 s, a pulse repetition frequency (PRF) of 100 Hz, a duty cycle of 10%, and an I_SPPA of 5 W/cm²). In this research, T1-, T2-w, and ZTE MRI scans with 1 mm isotropic resolution were obtained from neurologically healthy subjects. TMS target...

Discussion

In this method, subject-specific simulations are performed to predict and assess possible thermal and mechanical effects resulting from TUS application to the brain. Data sets between participants must remain separate and carefully documented, as using an incorrect scan or data file will lead to inaccurate simulations. When numerous participant scans are collected, and planning is performed together, it is important to ensure proper labeling of images and folders and proceed with caution when sorting and saving files.

Materials

Name	Company	Catalog Number	Comments
128-channel amplifier unit	Image Guided Therapy		This unit drives the H-317 transducer
24-channel head coil	General Electric
3D printer	Raise3D	Pro2	Filament thickness of 1.75mm.
3T MRI scanner	General Electric	Discovery 750 HD	MR Console version DV26.0_R05_2008
BabelBrain	Samuel Pichardo (University of Calgary)	Version 0.3.0	Accessible at https://github.com/ProteusMRIgHIFU/BabelBrain. Executes thermal and acoustic simulations.
Blender	Blender Foundation	Version 3.4.1	Accessible at https://www.blender.org. Blender is called automatically by BabelBrain.
Brainsight	Rogue Research	Version 2.5.2	Used for target identification, trajectory planning, and execution of TUS delivery sessions.
Chair and chin/head holder	Rogue Research		To be used during TUS delivery session to ensure stability of participant’s head for optimized targeting.
Custom-made coupling cone	University of Calgary team		3D printed cone in acrylonitrile butadiene styrene (ABS), only required for H-317 transducer.
dcm2niix	Chris Rorden (University of South Carolina)	Version 1.0.20220720	Accessible at https://github.com/rordenlab/dcm2niix/releases. Used for pre-processing subject MR images.
Fiducials and headband or glasses	Brainsight, Rogue Research	ST-1325 (subject tracker), LCT-583 (large coil tracker)	Headband or glasses can be interchangeably used.
Headphones	Beats	Fit Pro True Wireless Earbuds	Wireless Bluetooth earbuds with disposable tips.
MacBookPro	Apple	M2 Max, 16”, 64GB RAM	Computer for completing trajectory planning and simulations
SimNIBS	Axel Thielscher (Technical University of Denmark)	Version 4.0.0	Accessible at https://simnibs.github.io/simnibs/build/html.index.html
Syringe(s)		10 mL, 60 mL	Used to add additional ultrasound gel to fill air pockets.
Transducer	Sonicconcepts	H-317	Other supported transducers include CTX_500 (NeuroFUS, Sonicconcepts), Single element, H-246 (Sonicconcepts), and Bsonix (Brainsonix)
Transducer film	Sonicconcepts	Polyurethane membrane	Interface between transducer and the subject
Ultrasound gel	Wavelength	Clear Ultrasound Gel	Coupling medium.
Windows Laptop	Acer	Aspire A717-71G, Intel Core i7-7700HQ, 16 GB RAM	System used to control 128-channel amplifier and generate sound through the headphones