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We present a method for simultaneously collecting fMRI and fNIRS signals from the same subjects with whole-head fNIRS coverage. The protocol has been tested with three young adults and can be adapted for data collection for developmental studies and clinical populations.
Functional near-infrared spectroscopy (fNIRS) is a portable neuroimaging methodology, more robust to motion and more cost-effective than functional magnetic resonance imaging (fMRI), which makes it highly suitable for conducting naturalistic studies of brain function and for use with developmental and clinical populations. Both fNIRS and fMRI methodologies detect changes in cerebral blood oxygenation during functional brain activation, and prior studies have shown high spatial and temporal correspondence between the two signals. There is, however, no quantitative comparison of the two signals collected simultaneously from the same subjects with whole-head fNIRS coverage. This comparison is necessary to comprehensively validate area-level activations and functional connectivity against the fMRI gold standard, which in turn has the potential to facilitate comparisons of the two signals across the lifespan. We address this gap by describing a protocol for simultaneous data collection of fMRI and fNIRS signals that: i) provides whole-head fNIRS coverage; ii) includes short-distance measurements for regression of the non-cortical, systemic physiological signal; and iii) implements two different methods for optode-to-scalp co-registration of fNIRS measurements. fMRI and fNIRS data from three subjects are presented, and recommendations for adapting the protocol to test developmental and clinical populations are discussed. The current setup with adults allows scanning sessions for an average of approximately 40 min, which includes both functional and structural scans. The protocol outlines the steps required to adapt the fNIRS equipment for use in the magnetic resonance (MR) environment, provides recommendations for both data recording and optode-to-scalp co-registration, and discusses potential modifications of the protocol to fit the specifics of the available MR-safe fNIRS system. Representative subject-specific responses from a flashing-checkerboard task illustrate the feasibility of the protocol to measure whole-head fNIRS signals in the MR environment. This protocol will be particularly relevant for researchers interested in validating fNIRS signals against fMRI across the lifespan.
Cognitive function has been studied in the adult human brain via functional magnetic resonance imaging (fMRI) for nearly three decades. Although fMRI provides high spatial resolution and both functional and structural images, it is often not practical for studies conducted in naturalistic contexts or for use with infants and clinical populations. These constraints substantially limit our understanding of brain function. An alternative to fMRI is the use of portable methodologies that are more cost-effective and robust to motion, such as functional near-infrared spectroscopy (fNIRS)1,2,3. fNIRS has been used with infants and young children to assess brain function across a range of cognitive domains, such as language development, processing of socially relevant information and object processing 4,5,6. fNIRS is also a neuroimaging modality especially suitable for testing clinical populations due to its potential for repeated testing and monitoring across ages7,8,9. Despite its wide applicability, there are no studies quantitatively comparing fMRI and fNIRS signals collected simultaneously from the same subjects with whole-head coverage. This comparison is necessary to comprehensively validate area-level activations and functional connectivity between regions of interest (ROIs) against the fMRI gold standard. Furthermore, establishing this inter-modality correspondence has the potential to enhance the interpretation of fNIRS when it is the only collected signal across both typical and atypical development.
Both fMRI and fNIRS signals detect changes in cerebral blood oxygenation (CBO) during functional brain activation10,11. fMRI relies on changes in electromagnetic fields and provides a high spatial resolution of CBO changes12. fNIRS, in contrast, measures absorption levels of near-infrared light using a series of light-emitting and light-detecting optodes2. Since fNIRS measures changes in absorption at different wavelengths, it can assess concentration changes in both oxy- and deoxyhemoglobin. Prior studies using simultaneous recordings of fMRI and fNIRS signals with a small number of optodes have shown that the two signals have high spatial and temporal correspondence10. There are strong correlations between blood-oxygen-level-dependent (BOLD) fMRI and optical measures11,13, with deoxyhemoglobin showing the highest correlation with the BOLD response, as reported by prior work comparing the temporal dynamics of the fNIRS and fMRI hemodynamic response functions (HRFs)14. These early studies implemented motor response paradigms (i.e., finger tapping) and used a limited number of optodes covering primary motor and premotor cortex areas. In the last decade, studies have expanded the focus to include a larger battery of cognitive tasks and resting-state sessions, although still using a limited number of optodes covering specific ROIs. These studies have shown that variability in fNIRS/fMRI correlations is dependent on the optode's distance from the scalp and the brain15. Furthermore, fNIRS can provide resting-state functional connectivity measures comparable to fMRI16,17.
The current protocol builds on prior work and addresses key limitations by i) providing whole-head fNIRS coverage, ii) including short-distance measurements for regression of non-cortical physiological signals, iii) implementing two different methods for optode-to-scalp co-registration of fNIRS measurements and iv) enabling assessment of the test-retest reliability of the signal across two independent sessions. This protocol for simultaneous data collection of fMRI and fNIRS signals was initially developed for testing young adults. However, one of the goals of the study was to create an experimental setup for collecting simultaneous fMRI/fNIRS signals that can be subsequently adapted for testing developmental populations. Therefore, the current protocol can also be used as a starting point for developing a protocol to test young children. In addition to using whole-head fNIRS coverage, the protocol also aims to incorporate recent advances in the field of fNIRS hardware, such as the inclusion of short-distance channels to measure the systemic physiological signal (i.e., vascular changes arising from noncortical sources, such as blood pressure, respiratory and cardiac signals)18,19 ;and the use of a 3D structure sensor for optode-to-scalp co-registration20. Although the focus of the present protocol is on the results of a visual flashing checkerboard task, the entire experiment includes two sessions with a mix of traditional block-task designs, resting-state sessions, and naturalistic movie-viewing paradigms.
The protocol describes the steps needed to adapt the fNIRS equipment for use in the MRI environment, including cap design, temporal alignment via trigger synchronization and phantom tests required before the start of data collection. As noted, the focus here is on the results of the flashing checkerboard task, but the overall procedure is not task-specific and can be appropriate for any number of experimental paradigms. The protocol further outlines the steps required during data collection, which include fNIRS cap placement and signal calibration, participant and experimental equipment setup, as well as post-experiment clean up and data storage. The protocol ends by providing an overview of the analytic pipelines specific for preprocessing fNIRS and fMRI data.
The research was approved by the Institutional Review Board (IRB) at Yale University. Informed consent was obtained for all subjects. Subjects had to pass MRI screening to ensure their safe participation. They were excluded if they had a history of serious medical or neurological disorder that would likely affect cognitive functioning (i.e., a neurocognitive or depressive disorder, trauma, schizophrenia, or obsessive-compulsive disorder).
NOTE: The current protocol uses a CW-NIRS device with 100 long-distance channels and 8 short-distance channels (32 laser diode sources, λ = 785/830 nm with average power of 20mW / wavelength, and 38 avalanche photodiode detectors) sampled at 1.95 Hz. MRI and fMRI scans were collected on a Siemens 3 Tesla Prisma scanner using a 20-channel head-coil. All data were collected at the Yale Brain Imaging Center (https://brainimaging.yale.edu/). System-specific modifications for collecting simultaneous fMRI and fNIRS data are noted throughout the protocol.
1. fNIRS equipment modifications and development for simultaneous data collection
NOTE: Steps 3 to 6 are specific to the NIRScoutXP system and may not apply to other fNIRS systems due to variation in the acquisition software and available phantoms for optode assessment.
Figure 1. Equipment for simultaneous data collection of fMRI and fNIRS measurements. (A) Pouch made of black, water repellent material to store vitamin E capsules sewn on the fNIRS cap adjacent to each optode. (B) MRI-safe bridge to hold the optical fibers above the floor so they can reach the participant's head during data collection. (C) Parallel port replicator that transmits pulses from the scanner to the fNIRS device. Please click here to view a larger version of this figure.
2. Experimental task design
3. fNIRS cap placement and signal calibration on testing day
NOTE: All steps below take place in the MRI control or consent rooms, unless otherwise noted.
Figure 2. Short-distance detectors and tools for fNIRS cap preparation. (A) Short-distance detector probes and rubber buffers to be attached to the fNIRS cap over frontal areas where there is minimal hair. (B) From left to right: Cable organizers to arrange the optical fibers into bundles, MRI-safe applicators to push away the hair during optode placement, and plastic tweezers to remove optodes from the cap if needed during NIRS cap setup to displace hair. Please click here to view a larger version of this figure.
Figure 3. 3D Structure sensor digitizer and fNIRS cap placement. (A) Experimenter using the 3D structure sensor digitizer to create a 3D model of the participant's head. Green stickers are used to identify fiducial locations. (B) Optical fibers inserted into the fNIRS cap on a participant's head and arranged into bundles using cable organizers before signal calibration. Please click here to view a larger version of this figure.
4. Participant setup
NOTE: The following steps are conducted in the MRI scanner room. The use of a respiratory belt and pulse oximeter is optional and needed only if researchers are interested in regressing out these signals from the fNIRS data22. The protocol uses a respiratory belt, which is part of the respiratory unit for the acquisition of the respiratory amplitude using a restraint belt. Similarly, the physiological pulse unit consists of an optical plethysmography sensor that allows the acquisition of the cardiac rhythm.
Figure 4. Participant set up in the MRI scanner. (A) Pillows inside the MR head coil used to support the participant's head and optical fibers arranged into bundles before participant set up. (B) Participant laying on the scanner bed with the fNIRS cap ready for testing. The top of the head-coil has not yet been placed over the participant's face. Please click here to view a larger version of this figure.
5. Scanner and fNIRS equipment setup prior to signal recording
6. Simultaneous signal recording
Figure 5. Flashing checkerboard paradigm as the experimental task. Participants viewed a black-and-white checkerboard pattern with white squares flashing eight times per second that alternated with a gray screen showing a white circle. As an attention check, participants were instructed to press a button with their right hand upon seeing a white circle appear in the middle of the screen. Upon pressing the button, the circle turns red. The task was completed in a single run comprised of 22 blocks in total: 11 flashing checkerboard blocks and 11 inter-trial-periods. Flashing checkerboard periods lasted for 10 s and inter-trial periods lasted for 20 s. Thus, the onset of the flashing checkerboard occurred every 30 s (0.033 Hz). Displays were generated by PsychoPy v2021.2.4 and projected onto the rear facing mirror on the top of the head coil via a 1080p DLP projection system. Participants completed one run of this task (~6 min). Please click here to view a larger version of this figure.
7. Post-experiment clean up and data storage
8. fMRI data preprocessing
NOTE: The fMRI data were preprocessed following the minimal preprocessing pipelines from the Human Connectome Project23 using QuNex24, an open-source software suite that supports data organization, preprocessing, quality assurance, and analyses across neuroimaging modalities. Detailed documentation on the specific settings and parameters for each of the steps highlighted below can be found on the QuNex website at https://qunex.yale.edu/. Main steps and parameters used to process the data are presented below.
9. fNIRS data preprocessing
NOTE: The fNIRS data were analyzed following best practices in fNIRS data analysis25 using NeuroDOT26, an open-source environment for analysis of optical data from raw light levels onto voxel-level maps of brain function, which are co-registered to the anatomy of a specific participant or an atlas. All steps described below can be performed with NeuroDOT. Additional documentation on the specific settings and parameters for each of the steps highlighted below can be found in the tutorials and scripts at https://github.com/WUSTL-ORL/NeuroDOT_Beta. Finally, optode-to-scalp registration requires obtaining the fNIRS optode coordinates relative to the underlying brain tissue, which can be done using a 3D digitizer or vitamin E capsules as fiducials if available. Both methods are described in this section and references to the relevant software packages are provided.
10. fMRI/fNIRS task-evoked data analyses
This section presents representative subject-specific responses for the flashing checkerboard task for both fMRI and fNIRS signals. First, representative raw fNIRS data and quality assessments are shown in Figure 6 and Figure 7 to illustrate the feasibility of the experimental setup to measure fNIRS signals in the MRI environment. A diagram of the whole head optode array and sensitivity profile is shown in Figure 8....
This protocol for simultaneous data collection of fMRI and fNIRS signals uses a whole-head fNIRS optode array and short-distance channels for measuring and regressing out the systemic non-cortical physiological signals. Critical steps in this protocol include modification and development of the fNIRS equipment for collecting fNIRS signals in the MRI environment. To the best of our knowledge, there is no turn-key commercial system that is fully optimized for capturing simultaneous fMRI and fNIRS measurements usi...
Publication fees for this article are sponsored by NIRx. The authors have nothing else to disclose.
This research was supported by the following funding sources: A NARSAD Young Investigator Award Grant from the Brain and Behavior Research Foundation (Grant #29736) (SSA), a Global Grand Challenges Grant from the Bill and Melinda Gates Foundation (Grant #INV-005792) (RNA) and a Discovery Fund Grant from the Department of Psychology at Yale University (RNA). The authors also wish to acknowledge Richard Watts (Yale Brain Imaging Center) for his support during data collection and Adam Eggebrecht, Ari Segel and Emma Speh (Washington University in St Louis) for their assistance in data analysis.
Name | Company | Catalog Number | Comments |
280 low-profile MRI-compatible grommets for NIRs caps | NIRx | GRM-LOP | |
4 128-position NIRS caps with 128x unpopulated slits in 10-5 layout | NIRx | CP-128-128S | Sizes: 52, 54, 56, 60 |
8 bundles of 4x detector fibers with low-profile tip; MRI-, MEG-, and TMS-compatible. | NIRx | DET-FBO- LOW | 10 m long |
8 bundles of 4x laser source fibers with MRI-compatible low-profile tip | NIRx | SRC-FBO- LAS-LOW | 10 m long |
Bundle set of 8 short-channel detectors with specialized ring grommets that fit to low-profile grommets | NIRx | DET-SHRT-SET | Splits a single detector into 8 short channels that may be placed anywhere on a single NIRS cap |
Magnetom 3T PRISMA | Siemens | N/A | 128 channel capacity, 64/32/20 channel head coils, 80 mT/m max gradient amplitude, 200 T/m/s slew rate, full neuro sequences |
NIRScout XP Core System Unit | NIRx | NSXP- CHS | Up to 64x Laser-2 (or 32x laser-4) illuminators or 64 LED-2 illuminators; up to 32x detectors; capable of tandem (multi-system) and hyperscanning (multi-subject) measurements; compatible with EEG, tDCS, eye-tracking, and other modalities; modules available for fMRI, TMS, MEG compatibility |
NIRStar software | NIRx | N/A | Version 15.3 |
NIRx parallel port replicator | NIRx | ACC-LPT-REP | The parallel prot replicator comes with three components: parallel port replicator box, USB power cable and BNC adapter |
Physiological pulse unit | Siemens | PPU098 | Optical plethysmography allowing the acquisiton of the cardiac rhythm. |
Respiratory unit | Siemens | PERU098 | Unit intended for the acquisition of the respiratory amplitude (by means of a pneumatic system and a restraint belt). |
Structure Sensor Mark II | Occipital | 101866 (SN) | 3D structure sensor for optode digitization. |
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