This work was supported in part by a Natural Sciences and Engineering Research Council of Canada Discovery Grant, the INNOVAIT program, Cumming Medical Research Fund, Canada Foundation for Innovation (Project 36703), Hotchkiss Brain Institute CAPRI Grant, and Parkinson Association of Alberta Funding. GBP acknowledges support from the Canadian Institutes for Health Research (FDN-143290) and the Campus Alberta Innovates Chair Program.

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 his...

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.</p...

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 precision1,2. By contrast, transcranial ultrasound (TUS) is an emerging non-invasive technique capable of enhancing or suppressing neuronal activity3,4,5 and targeting cortical or subcortical structures at millimeter precision6,7. Animal models using rodents4,8,9, rabbits10, sheep5,11, swine6, and nonhuman primates7,12,13,14 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)&#160;and performance on a reaction time task when targeting the primary motor cortex15,16 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, sheep5,11, 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 (Isppa) 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 humans20,21,22,23,24. Although the mechanisms of TUS neuromodulation have been proposed to mainly involve mechanical agitation of lipid membranes leading to the opening of ion channels25,26,27, 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 application28,29. 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 TUS32,33,34 in animals and humans, whereby TUS stimulation can activate auditory pathways in the brain to elicit responses32,33,34. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>128-channel amplifier unit</td>
			<td>Image Guided Therapy</td>
			<td></td>
			<td>This unit drives the H-317 transducer</td>
		</tr>
		<tr>
			<td>24-channel head coil</td>
			<td>General Electric</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Raise3D</td>
			<td>Pro2</td>
			<td>Filament thickness of 1.75mm.</td>
		</tr>
		<tr>
			<td>3T MRI scanner</td>
			<td>General Electric</td>
			<td>Discovery 750 HD</td>
			<td>MR Console version DV26.0_R05_2008</td>
		</tr>
		<tr>
			<td>BabelBrain</td>
			<td>Samuel Pichardo (University of Calgary)</td>
			<td>Version 0.3.0</td>
			<td>Accessible at https://github.com/ProteusMRIgHIFU/BabelBrain. Executes thermal and acoustic simulations.</td>
		</tr>
		<tr>
			<td>Blender</td>
			<td>Blender Foundation</td>
			<td>Version 3.4.1</td>
			<td>Accessible at https://www.blender.org. Blender is called automatically by BabelBrain.</td>
		</tr>
		<tr>
			<td>Brainsight</td>
			<td>Rogue Research</td>
			<td>Version 2.5.2</td>
			<td>Used for target identification, trajectory planning, and execution of TUS delivery sessions.</td>
		</tr>
		<tr>
			<td>Chair and chin/head holder</td>
			<td>Rogue Research</td>
			<td></td>
			<td>To be used during TUS delivery session to ensure stability of participant&#8217;s head for optimized targeting.</td>
		</tr>
		<tr>
			<td>Custom-made coupling cone</td>
			<td>University of Calgary team</td>
			<td></td>
			<td>3D printed cone in acrylonitrile butadiene styrene (ABS), only required for H-317 transducer.</td>
		</tr>
		<tr>
			<td>dcm2niix</td>
			<td>Chris Rorden (University of South Carolina)&#160;</td>
			<td>Version 1.0.20220720</td>
			<td>Accessible at https://github.com/rordenlab/dcm2niix/releases. Used for pre-processing subject MR images.</td>
		</tr>
		<tr>
			<td>Fiducials and headband or glasses</td>
			<td>Brainsight, Rogue Research&#160;</td>
			<td>ST-1325 (subject tracker), LCT-583 (large coil tracker)</td>
			<td>Headband or glasses can be interchangeably used.</td>
		</tr>
		<tr>
			<td>Headphones</td>
			<td>Beats</td>
			<td>Fit Pro True Wireless Earbuds</td>
			<td>Wireless Bluetooth earbuds with disposable tips.</td>
		</tr>
		<tr>
			<td>MacBookPro</td>
			<td>Apple</td>
			<td>M2 Max, 16&#8221;, 64GB RAM</td>
			<td>Computer for completing trajectory planning and simulations</td>
		</tr>
		<tr>
			<td>SimNIBS</td>
			<td>Axel Thielscher (Technical University of Denmark)</td>
			<td>Version 4.0.0</td>
			<td>Accessible at https://simnibs.github.io/simnibs/build/html.index.html</td>
		</tr>
		<tr>
			<td>Syringe(s)</td>
			<td></td>
			<td>10 mL, 60 mL</td>
			<td>Used to add additional ultrasound gel to fill air pockets.</td>
		</tr>
		<tr>
			<td>Transducer</td>
			<td>Sonicconcepts</td>
			<td>H-317</td>
			<td>Other supported transducers include CTX_500 (NeuroFUS, Sonicconcepts), Single element, H-246 (Sonicconcepts), and Bsonix (Brainsonix)</td>
		</tr>
		<tr>
			<td>Transducer film</td>
			<td>Sonicconcepts</td>
			<td>Polyurethane membrane</td>
			<td>Interface between transducer and the subject</td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Wavelength</td>
			<td>Clear Ultrasound Gel</td>
			<td>Coupling medium.</td>
		</tr>
		<tr>
			<td>Windows Laptop</td>
			<td>Acer</td>
			<td>Aspire A717-71G, Intel Core i7-7700HQ, 16 GB RAM</td>
			<td>System used to control 128-channel amplifier and generate sound through the headphones</td>
		</tr>
	</tbody>
</table>

pipeline for planning and execution of transcranial ultrasound neuromodulation experiments in humans

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.

Figure 7&#160;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 ISPPA of 5 W/cm&#178;). In this research, T1-, T2-w, and ZTE MRI scans with 1 mm isotropic resolution were obtained from neurologically healthy subjects. TMS target...

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.

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

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation technique capable of manipulating both cortical and subcortical ...