A Methodological Protocol and Considerations for Transcranial Ultrasonic Stimulation in Exploratory Clinical Human Studies

Summary

Transcranial ultrasonic stimulation (TUS) is a promising non-invasive technique capable of stimulating the human brain at any depth, offering new therapeutic possibilities to treat various neurological conditions. This protocol provides a standardized yet adaptable empirical framework for applying TUS in neurotypical adults and patients with neurological diseases such as stroke.

Abstract

Transcranial ultrasonic stimulation (TUS) is emerging as a non-invasive neuromodulatory technique capable of delivering millimeter-precision stimulation at whole-brain depths. Research efforts have increasingly focused on its translational potential. Promising data have been reported across several disease populations, including Parkinson's disease and stroke, paving the way for clinical applications of TUS. Clinical studies to date, however, show substantial variability in transducer fixation, targeting approaches, and acoustic parameters. This limits the interpretability and comparability of results. Existing methodological guides address human TUS in general but do not focus on applications in neurological populations. This experimental protocol presents a standardized yet adaptable framework for applying TUS to neurological cohorts such as stroke. It offers detailed guidance on: (1) essential and optional hardware components in the context of therapy-oriented TUS; (2) hardware settings and parameter selection, including strategies to minimize auditory confounds; (3) calibration and quality assurance procedures to ensure the transducer delivers waveforms as specified; (4) targeting approaches based on simulation or non-simulation methods for accurate localization of TUS focus/foci to the intended anatomical region(s); (5) methodology adaption for clinical populations; and (6) outcome measures for clinical TUS, encompassing safety assessments and surrogate outcome measures such as corticospinal excitability and motor sequence learning. This protocol is designed as a replicable, modular resource. It accommodates both novice users (seeking a practical entry point into patient-based TUS) and experienced researchers (aiming to align with emerging scientific and methodological standards). The goal is to support the growing clinical interest in TUS and to facilitate clinically translatable, reproducible, and comparable results across research groups and patient populations.

Introduction

A unique and desirable combination, the deep, focal, and non-invasive nature of transcranial ultrasonic stimulation¹ (TUS) has spurred growing interests in exploring its therapeutic potentials for neurological and neuropsychiatric diseases (e.g., stroke², Parkinson's disease³, depression⁴, Alzheimer's disease⁵, etc.). Expert consensus and guidelines have sought to improve the reproducibility and interpretability of TUS research^6,7. However, these primarily address the broader basic research a....

Protocol

Experimental procedures presented in Huang et al., 2025² have been approved by the Duke Institutional Review Board and are in accordance with the recently updated Declaration of Helsinki. Obtain informed consent from each participant before enrollment.

NOTE: Follow the instructions while simultaneously working with the hardware/software to enhance understanding and ensure accurate implementation. Figure 3A shows an overview of the steps involved, Figure 3B shows a decision flowchart for choosing simulation-based (Step 3) versus non-simulation-based (Step 4) ....

Results

Simulation-based TUS targeting first defines an initial transducer placement manually based on proximity to the intracranial target and perpendicularity to the skull and uses this initial placement and transducer specifications to solve for the intracranial acoustic field. Next, it computes the discrepancy between the intended stimulation target and the acoustic field focus, updates the transducer placement in the reverse direction of this discrepancy, solves for the acoustic field using this new transducer placement, an.......

Discussion

There has been significant research enthusiasm for the application of TUS in neurological conditions; however, a need exists for increased scientific rigor and replication of results. A successful TUS on the intended target tissue relies on several critical steps. First, the transmitting sensitivity of the transducer needs to be correctly characterized. Options include following Step 2 of this protocol, outsourcing to a collaborator, and utilizing vendor's service. The second critical step is targeting, i.e., determining.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3D printer	UltiMaker	S3	To be used to manufacture the transducer mount.
3T MRI scanner	General Electric	Signa UHP
BabelBrain	Samuel Pichardo (University of Calgary)	Version 0.4.3	Referred in main text as the "TUS targeting software." Accessible at https://proteusmrighifu.github.io/BabelBrain/
Brainsight	Rogue Research		Referred in main text as the "neuronavigation software/system." To be used for TUS/TMS targeting, comes with a Mac computer, an infrared camera, and fiducials.
EMG amplifier and analog-to-digital converter	Cambridge Electronic Design Ltd	CED 1902,  MICRO4	To be used to acquire, amplify, and digitize EMG signals.
dcm2niix	Christopher Rorden	V1.0.20250506	To convert neuroimages from DICOM to NIFTI. Accessible at https://pypi.org/project/dcm2niix/
E-prime	Psychology Software Tools	E-prime 3.0	Referred in main text as an example software for MSL. To be used to create the discrete sequence production task.
Fiducials and headband	Rogue Research	ST-1275 (subject tracker), LCT-494 (large coil tracker), P-1528 (pointer)	To be used in conjunction with Brainsight for neuronavigation.
FSL	University of Oxford		Referred in main text as the "DTI processing software." To be used in the optional but recommended Step 8 for diffusion image processing.
Function generator	Keysight Technologies	33210A	To generate the drive signal for the TUS transducer.
Hydrophone	Onda Corporation	HNA-0400	To measure pressure output from the transducer.
ITK-SNAP	Penn Image Computing and Science Laboratory	ITK-SNAP 4.0	Referred in main text as an "MRI viewer." Accessible at https://www.itksnap.org/pmwiki/pmwiki.php?n=Downloads.SNAP4
k-Wave	Bradley Treeby, Ben Cox, and Jiri Jaros	k-Wave Version 1.4	Referred in main text as the "open-source acoustic simulation software." Accessible at http://www.k-wave.org/
MATLAB	MathWorks Inc.	R2024b	Referred in main text as the "programming platform." Accessible at https://www.mathworks.com/products/matlab.html
MRIcroGL	NeuroImaging Tools & Resources Collaboratory	v1.2.20220720	To convert neuroimages from DICOM to NIFTI. Accessible at https://www.nitrc.org/projects/mricrogl/
MRIcron	NeuroImaging Tools & Resources Collaboratory	v1.0.20190902	Referred in main text as an "MRI viewer." Accessible at https://www.nitrc.org/projects/mricron
Oscilloscope	SIGLENT Technologies	SDS 1202X-E	To be used to measure the input of TUS transducer and to record hydrophone measurements.
Python	Python Software Foundation	Python 3.13	Referred in main text as an example language for MSL. To be used to create the discrete sequence production task.
RF power amplifier	AR RF/Microwave Instrumentation	50A250	To be used to amplifier the output of function generators to the amplitude required by the TUS transducer.
Signal	Cambridge Electronic Design Ltd	Signal 7.05a (x86)	Referred in main text as the "EMG acquisition software." To be used to acquire EMG signals from the analog-to-digital converter to a computer.
SimNIBS	Axel Thielscher (Technical University of Denmark)	Version 4.5	Referred in main text as the "e-field software." Accessible at https://simnibs.github.io/simnibs/build/html/index.html
SPM12	Functional Imaging Laboratory (University College London)		Referred in main text as the "MRI processing software." To be used in the optional but recommended Step 8 for MRI image processing.
TMS stimulator and coil	Magstim Inc.	BiStim2, 70 mm figure-of-eight	To be used to apply TMS pulses.
Transducer	Blatek Industries	AT32080
Transducer mount	Duke University team		3D printed tranducer mount in polyactic acid (PLA) and added with fabric straps.
Ultrasound gel	Parker Laboratories	Aquasonic 100	To be applied between the TUS transducer and participant scalp.
Water tank and 3-axis positioning system	Onda Corporation	AIMS III Hydrophone Scanning System	Customized with the company.