This work is supported by the ECNU Academic Innovation Promotion Program for Excellent Doctoral Students (YBNLTS2019-025) and the National Natural Science Foundation of China (31872783 and 71942001).

All recruited participants (40 dyads, mean age 22.1 &#177; 1.2 years; 100% right-handed; normal or corrected-to-normal vision) were healthy.&#160;Before the experiment, participants gave informed consent. Participants were financially compensated for their participation. The study was approved by the University Committee of Human Research Protection (HR-0053-2021), East China Normal University.
1. Preparation steps before adopting data
<ol>
	<li>Homemade NIRS caps
	<ol>
		<li>Adopt elastic s...

First, in the present protocol, the specific steps of conducting fNIRS hyperscanning experiments in a collaborative learning scenario are stated. Second, the data analysis pipeline that assesses the IBS of hemodynamic signals in collaborative learning dyads is also presented. The detailed operation on conducting fNIRS hyperscanning experiments would promote the development of open-science. Furthermore, the analysis pipeline is provided here to increase the reproducibility of hyperscanning research. In the following, the ...

Recently, to reveal the concurrent brain activity across the interactive dyads or members of a group, researchers employ the hyperscanning approach1,2. Specifically, electroencephalogram (EEG), functional magnetic resonance imaging (fMRI), and functional near-infrared spectroscopy (fNIRS) are used to record the neural and brain activities from two or more subjects simultaneously3,4,5. Researchers extract a neural index entailing concurrent brain coupling based on this technique, which refers to inter-brain synchrony (IBS) (i.e., phase and/or amplitude alignment of the neuronal or hemodynamic signals across time). A large variety of hyperscanning research found IBS during social interaction between multiple individuals (e.g., player-audience, instructor-learner, and leader-follower)6,7,8. Furthermore, IBS holds specific implications of effective learning and instruction9,10,11,12,13,14. With the surging of hyperscanning research in naturalistic learning scenarios, establishing a standard protocol of hyperscanning experiments and the pipeline of data analysis in this field is necessary.
Thus, this paper provides a protocol for conducting fNIRS-based hyperscanning of collaborative learning dyads and a pipeline for analyzing IBS. fNIRS is an optical imaging tool, which radiates near-infrared light to assess the spectral absorption of hemoglobin indirectly, and then hemodynamic/oxygenation activity is measured15,16,17. Compared with fMRI, fNIRS is less prone to motion artifacts, allowing measurements from subjects who are doing real-life experiments (e.g., imitation, talking, and non-verbal communication)18,7,19. In comparison with EEG, fNIRS holds higher spatial resolution, allowing researchers to detect the location of brain activity20. Thus, these advantages in spatial resolution, logistics, and feasibility qualify fNIRS to conduct hyperscanning measurement1. Using this technology, an emerging research body detects an index term as IBS-the neural alignment of two (or more) people&#39;s brain activity-in different forms of naturalistic social settings9,10,11,12,13,14. In those studies, various methods (i.e., Correlation analysis and Wavelet Transform Coherence (WTC) analysis) are applied to calculate this index; meanwhile, a standard pipeline on such analysis is essential but lacking. As a result, a protocol for conducting fNIRS-based hyperscanning and a pipeline using WTC analysis to identify IBS is presented in this work
This study aims to evaluate IBS in collaborative learning dyads using the fNIRS hyperscanning technique. First, a hemodynamic response is recorded simultaneously in each dyads&#39; prefrontal and left temporoparietal regions during a collaborative learning task. These regions have been identified as associated with interactive teaching and learning9,10,11,12,13,14. Second, the IBS is calculated on each corresponding channel. The fNIRS data recording process consists of two parts: resting-state session and collaborative session. The resting-state session lasts for 5 min, during which both the participants (sitting face-to-face, apart from one another by a table (0.8 m)) are required to remain still and relax. This resting-state session is served as the baseline. Then, in the collaborative session, the participants are told to study the entire learning materials together, eliciting understanding, summarizing the rules, and making sure all learning materials are mastered. Here, the specific steps of conducting the experiment and fNIRS data analysis are presented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EEG caps</td>
			<td>Compumedics Neuroscan,Charlotte,USA</td>
			<td>64-channel Quik-Cap</td>
			<td>We choose two sizes of cap(i.e.medium and large).</td>
		</tr>
		<tr>
			<td>NIRS measurement system with probe sets and probe holder grids</td>
			<td>Hitachi Medical Corporation, Tokyo, Japan</td>
			<td>ETG-7100 Optical Topography System</td>
			<td>The current study protocol requires an optional second adult probe set for 92 channels of measurement in total.</td>
		</tr>
		<tr>
			<td>Numeric computing platform</td>
			<td>The MathWorks, Inc., Natick, MA</td>
			<td>MATLAB R2020a</td>
			<td>Serves as base for Psychophysics Toolbox extensions (stimulus presentation), Homer2&#160; (fNIRS preprocess analysis), and &#34;wtc&#34; function(WTC computation).</td>
		</tr>
		<tr>
			<td>Psychology software</td>
			<td>psychology software tools,Sharpsburg, PA,USA</td>
			<td>E-prime 2.0</td>
			<td>we apply E-prime to start the fNIRS measurement system and send triggers which marking the rest phase and collaborative learning phase for fNIRS recording data</td>
		</tr>
		<tr>
			<td>Swimming caps</td>
			<td>Zoke corporation,Shanghai,China</td>
			<td>611503314</td>
			<td>We first placed the standard 10-20 EEG cap on the head mold, and placed the swimming cap on the EEG cap. Second, we marked (inion, Cz, T3, T4, PFC and P5) with chalk.</td>
		</tr>
		<tr>
			<td>Three-dimensional (3-D) digitizer</td>
			<td>Polhemus, Colchester, VT, USA;</td>
			<td>Three-dimensional (3-D) digitizer</td>
			<td>Anatomical locations of optodes in relation to standard head landmarks were determined for each participant using a Patriot 3D Digitizer</td>
		</tr>
	</tbody>
</table>

inter-brain synchrony in open-ended collaborative learning: an fnirs-hyperscanning study

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the concurrent brain activity of two or more interactive individuals with a novel index called inter-brain synchrony (IBS) (i.e., phase and/or amplitude alignment of the neuronal or hemodynamic signals across time). A protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is presented here. Further, a pipeline of analyzing IBS of oxygenated hemoglobin (Oxy-Hb) signal is explained. Specifically, the experimental design, the process of NIRS data recording, data analysis methods, and future directions are all discussed. Overall, implementing a standardized fNIRS hyperscanning pipeline is a fundamental part of second-person neuroscience. Also, this is in line with the call for open-science to aid the reproducibility of research.

Figure 1 illustrates the experimental protocol and probe location. The fNIRS data recording process consists of two parts: resting-state session (5 min) and collaborative session (15-20 min). The collaborative learning dyads are required to relax and to keep still in the resting-state session. After that, participants are told to co-learning the learning material (Figure 1A). Their prefrontal and left temporoparietal regions are covered by the corresponding prob...

Watch this Scientific Journal Video about Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study at JoVE.com

Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study

The protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is outlined. Further, a pipeline to analyze the Inter-Brain Synchrony (IBS) of oxygenated hemoglobin (Oxy-Hb) signals is presented.

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the ...

This protocol reveals inter-brain synchrony in the collaborative dyads in the naturalistic environment by the fNIRS hyperscanning technique. The significant advantage of this technique is it allows investigation of real-time dynamics of two or more interacting brains. Before beginning the experiment, start the near infrared spectroscopy system for achieving a stable operating temperature, then adopt an elastic swimming cap to place the optode-holder grid and prepare the NIRS caps as described in the text manuscript.For a channel with insufficient signals, use the lighted fiber optic probes to move the hair under the probe's tip to one side. Once the operating temperature stabilizes, ensure that the trigger receiver is active and set the measurement mode to event-related measurement. Prepare the lighted fiber optic probe to move hair aside.Instruct the participants regarding the details of NIRS measurement methods and not to look directly into the laser beams as the laser beam may be harmful to the participant's eyes. Make the participants sit face to face to make sure they can see each other directly and adjust the chair-to-table distance to make the participants sit comfortably. Turn on the laser button, put the four optical fiber bundles loosely on the holder's arms without contact with the participants or chairs and place the cap with the probe sets on the participants'heads, ensuring that the three-by-five probe sets cover the forehead of the participant and the four-by-four probe sets cover the left temporoparietal cortex, then carefully push each spring-load probe further into the probe socket so that the probe's tip touches the scalp of the participant.Then allow two participants to co-learn the learning materials with five minutes rest state, which serves as the baseline. First, to check the quality of the signal, click the auto gain in the probe set monitor window of the functional NIRS machine, and then mark the channel's poor signal in yellow and the sufficient signal in green and repeat this procedure until all the channels are marked green. After the experiment, click on the Text File Out to export the raw light intensity data and save the data as a text file.Use the three-dimensional digitizer to determine the locations of emitters, receivers, and other references for each participant, then obtain the MNI coordinates of the recording channels using the commercially available numeric computing platform. The data from each participant in each channel were analyzed. The optodensity in channel 33 for a certain dyad was visualized.The data were extracted using the wavelet-based motion artifacts removing method, alone and along with the principal components analysis. The difference between the curves suggests that PCA is efficient in removing non-neural signals. The wavelet transform coherence matrix was visualized.The color map varied from blue to yellow, representing inter-brain synchrony values ranging from zero to one, where one denotes the largest coherence between two signals and zero denotes no coherence. In addition, significant coefficients were also obtained, the strong coherence around one hertz representing the dyad's cardiac rhythm coherence. The comparison between the observed T value and the distribution of random T value shows significant results in identified FOI.It's important to make sure the probe set covers the region of interests and try to reduce the deviation.

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JoVE Journal

Neuroscience

3.9K visualizações.  East China Normal University. Este protocolo revela a sincronia inter-cerebral nos díades colaborativos no ambiente naturalista pela técnica de hiperscanagem fNIRS. A vantagem significativa dessa técnica é que permite a investigação da dinâmica em tempo real de dois ou mais cérebros interagindo. Antes de iniciar o experimento, inicie o sistema de espectroscopia infravermelha próxima para alcançar uma temperatura de operação estável, em seguida, adote uma tampa de natação elástica para colocar a grade porta...