**2. NIRS and fundamentals of measurements**

For the evaluation of rehabilitative activity that involve prefrontal cortex (PFC), we used the NIRS functional detector systems: near-infrared spectroscopy uses low-intensity optical radiations to measure changes in light absorption by the cortical vascular tissues in order to detect changes in local concentration of oxy- and deoxyhemoglobin as a correlate of functional brain activity.

Each measurement channel is formed by an optic emitter (source) and a receiver (detector) placed on the subject's head. Due to the scattering (light diffusion) properties of the tissue, a portion of the received light will deeply pierce in the tissue structure, where it interacts with chromophores like hemoglobin. The degree of penetration and the shape of the probing volume are determined by the source-detector distance and by the optical properties of local tissues. Cortical NIRS signals are estimated to originate from an area that is placed between source and detector and from a tissue depth that is no more than the half of the source-detector distance (see **Figure 1**). Optical signals are weakened by biological tissues; intensity will decrease in terms of centimeters, therefore the optimal distance between source and detector is a compromise that must be verified in order to reach the maximum depth while maintaining a sufficient signal quality (signal-to-noise ratio). The optimal distance used in this study is the verified one of 30 mm and the cortical detection depth is around 25 mm.

This study focuses on NIRS ability to express suitable measures for the most adequate rehabilitation process with results that permit to evaluate the effects of PFC activation over emotions. Specifically, the neural mechanism underlying these processes is based on a bidirectional interaction between emotion and action, excellent to be measured using NIRS technique.

The Cerebro procedure aims at setting the fundamentals for NIRS application in those fields whose aim is to evaluate suitable rehabilitation models in prefrontal cortex related disorders by analyzing task-related stimuli that are mediated by pathways involving sensory processing, memory and emotion. To do so, NIRS parameters during recording will be considered along with the NIRS level of criticality in order to determine the good practice for functional

When talking about prefrontal cortex (PFC), we need to remember that the associative cortex in the frontal lobe has a lagging development in the neocortical regions. PFC is one of the cortical regions which undergoes a major expansion during personal maturation and evolution. In human adults, PFC represents approximately one-third of the whole neocortex. The PFC

In developing brains, like infants', PFC's delayed maturation is marked by a late myelination

Prefrontal cortex in primates is necessary not only for keeping relevant information in mind to complete a task but also for the active suppression of the irrelevant stimuli. Patients with ictus or pathological aging, affecting the lateral portion of the PFC, can discriminate between

This information is important in order to define the NIRS-fNIRS ratio (fNIRS: functional nearinfrared spectroscopy since this is used when a stimulus is given, and the outcome is a measurement of how the stimulus affects brain activity; if not it is a resting state registration that we call NIRS) where inhibitory responses or noise can influence the assessment of the result. Noise is the key point for the identification of an optimal functional assessment; therefore, in the following paragraph, we focus on specific measurements, precautions to be taken and the

For the evaluation of rehabilitative activity that involve prefrontal cortex (PFC), we used the NIRS functional detector systems: near-infrared spectroscopy uses low-intensity optical radiations to measure changes in light absorption by the cortical vascular tissues in order to detect changes in local concentration of oxy- and deoxyhemoglobin as a correlate of functional brain

Each measurement channel is formed by an optic emitter (source) and a receiver (detector) placed on the subject's head. Due to the scattering (light diffusion) properties of the tissue, a portion of the received light will deeply pierce in the tissue structure, where it interacts with chromophores like hemoglobin. The degree of penetration and the shape

ongoing and wide development is demonstrated by its impeccable structure.

auditory tasks but fail when irrelevant auditory stimuli are included [1].

right procedure to follow, step by step, in the preliminary phase of testing.

**2. NIRS and fundamentals of measurements**

processing of the required result.

of axonal connections.

86 Prefrontal Cortex

activity.

To achieve spatial imaging of the PFC, we used the NIRX imaging equipment that employs matrixes of paired source-detectors arranged on the area of interest, where each source channel forms a measurement channel with each detector channel. Therefore, a setup with X sources (S) and Y detectors (D) will produce an X\*Y measurement channel. This is true despite the position and the source-detector distance although only those channels whose distance is within a certain limit will produce signals with usable amplitudes and noise levels. Due to this model, there are no restrictions on how to set up sources and detectors, thus allowing us the maximum flexibility and freedom in realizing this functional study. At the same time, we focused on the experimental design ensuring signal quality during the set up and consequent optimal signal values data analysis. Meanwhile, we aimed at a perfect positioning which could guarantee signal quality and consequently adequate data analysis.

In order to achieve the abovementioned goals, calibration procedures were made for each functional assessment in which each source-detector combination was optimized. According to the quantity of light emitted by the source and received by the detector, the system predicts the optimal amplification signal, that is, the best signal quality that can be achieved. This process is automatically run by the control system during the calibration phase.

**Figure 1.** (Courtesy of NIRx Medical Technologies) Each measurement channel is formed by an optic emitter (source) and a receiver (detector) placed on the tissue surface. Cortical NIRS signals are estimated to originate from an area that is placed between source and detector and from a tissue depth that is no more than half of the source-detector distance.

One-year effort was needed in order to gain familiarity and competence with this system, and this amount of time is largely recommended in order to avoid hardware failure resulting in variations of the identified values.

**Table 1** shows signals criterion that are considered from right to left; algorithm quality scale checks if optimal level is reached for each channel, then evaluates signal strengths for each wavelength (760–850 nm) and estimates the detected noise level. The final signal quality clas-

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**Figure 3** shows a signal quality map where every channel has been judged to be "excellent" (in green) or "acceptable" (in yellow); this means that each channel achieved amplification gain intervals from 1 to 6. Levels ranged from 0.09 to 1.40 and the corresponding noise level is

The button "Refresh" allows us to update quality assessment without running a new calibration. It is important to keep in mind that a quick and well-done montage setup will shorten this calibration phase in order not to stress out the subject emotionally. The topographic layout helps to locate each channel and to act on a specific channel to set optimal signal quality.

**Table 1.** (Courtesy of NIRx Medical Technologies) shows signals criterion that are considered from right to left; the final

**Figure 3.** (Courtesy of NIRx Medical Technologies) shows a topographic layout in which excellent signal quality are

sification for a given channel depends on the worst marker obtained.

<2.5%. These values are shown in an appropriate software window.

signal quality classification for a given channel depends on the worst marker obtained.

displayed for each channel except for one that is acceptable.

The NIRS model used in this study is named NIRSport (**Figure 2**, 8 × 8 imaging system (8 × 8 mean 8 sources and 8 detectors). This NIRS hardware is attached to a pre-configured tablet or PC throughout a USB 2.0 cable. Every NIRS montage cap, on which sources and detectors are attached, follows the 128 standard EEG positions (known as the 10/20 international system).

### **2.1. Optimal system check**

Before running the system, sources and detectors must be placed on subject's head. The montage, in this case the Prefrontal Cortex (PFC) one, was chosen among a wide selection of different montages provided by NIRX. By starting the program, a system data sheet is displayed with a typical configuration setup for data acquisition.

Signal quality is the pre-requisite for a good functional recording; thus, before starting to record, it is necessary to run a source-detector calibration to verify it.

NIRS provides quality signal for each channel, classified by color (**Table 1**):

• Excellent (green): this quality level allows a clear view of heart fluctuation in the HBO signal and is appropriate for highest demands such as single-trial/single-subject evaluation. Cardiac signal may not be discernible on the display due to physiological reasons, but the output allows to detect the current noise and pull out neural activity with a suitable statistical analysis (filtering, SPM event-related mean, group mean).

If signal quality is low (red or white), neural activation may not be visible and blocked by noise signal. The most probable reason is the optical quality of the tissue itself. Losing the signal and consequently losing a channel information data are mostly due to the erroneous location of sources and detectors.

**Figure 2.** (Courtesy of NIRx Medical Technologies) NIRSport 8 × 8 imaging system; detectors and sources must be placed in the corresponding slot, equally for triggers sent to the software through a specific equipment (if necessary), the connection USB cable and, on the other side, the power supply connector.

**Table 1** shows signals criterion that are considered from right to left; algorithm quality scale checks if optimal level is reached for each channel, then evaluates signal strengths for each wavelength (760–850 nm) and estimates the detected noise level. The final signal quality classification for a given channel depends on the worst marker obtained.

One-year effort was needed in order to gain familiarity and competence with this system, and this amount of time is largely recommended in order to avoid hardware failure resulting in

The NIRS model used in this study is named NIRSport (**Figure 2**, 8 × 8 imaging system (8 × 8 mean 8 sources and 8 detectors). This NIRS hardware is attached to a pre-configured tablet or PC throughout a USB 2.0 cable. Every NIRS montage cap, on which sources and detectors are attached, follows the 128 standard EEG positions (known as the 10/20 international system).

Before running the system, sources and detectors must be placed on subject's head. The montage, in this case the Prefrontal Cortex (PFC) one, was chosen among a wide selection of different montages provided by NIRX. By starting the program, a system data sheet is displayed

Signal quality is the pre-requisite for a good functional recording; thus, before starting to

• Excellent (green): this quality level allows a clear view of heart fluctuation in the HBO signal and is appropriate for highest demands such as single-trial/single-subject evaluation. Cardiac signal may not be discernible on the display due to physiological reasons, but the output allows to detect the current noise and pull out neural activity with a suitable

If signal quality is low (red or white), neural activation may not be visible and blocked by noise signal. The most probable reason is the optical quality of the tissue itself. Losing the signal and consequently losing a channel information data are mostly due to the erroneous

**Figure 2.** (Courtesy of NIRx Medical Technologies) NIRSport 8 × 8 imaging system; detectors and sources must be placed in the corresponding slot, equally for triggers sent to the software through a specific equipment (if necessary), the

connection USB cable and, on the other side, the power supply connector.

variations of the identified values.

88 Prefrontal Cortex

**2.1. Optimal system check**

location of sources and detectors.

with a typical configuration setup for data acquisition.

record, it is necessary to run a source-detector calibration to verify it.

NIRS provides quality signal for each channel, classified by color (**Table 1**):

statistical analysis (filtering, SPM event-related mean, group mean).

**Figure 3** shows a signal quality map where every channel has been judged to be "excellent" (in green) or "acceptable" (in yellow); this means that each channel achieved amplification gain intervals from 1 to 6. Levels ranged from 0.09 to 1.40 and the corresponding noise level is <2.5%. These values are shown in an appropriate software window.

The button "Refresh" allows us to update quality assessment without running a new calibration. It is important to keep in mind that a quick and well-done montage setup will shorten this calibration phase in order not to stress out the subject emotionally. The topographic layout helps to locate each channel and to act on a specific channel to set optimal signal quality.


**Table 1.** (Courtesy of NIRx Medical Technologies) shows signals criterion that are considered from right to left; the final signal quality classification for a given channel depends on the worst marker obtained.

**Figure 3.** (Courtesy of NIRx Medical Technologies) shows a topographic layout in which excellent signal quality are displayed for each channel except for one that is acceptable.

By clicking on the "Details" button, a detailed calibration window will open with individual maps of gain, level and noise. Gain map: an inverse relationship exists between gain and intensity of the received light, intensity of light must be more amplified if the light signal is weak. If the outdistance between sources and detectors is between 2.5 and 3.0 cm, we expect a gain of 4, if lower than 4 there is a low level of light attenuation. If channels do not reach a maximum gain level of 7, the signal levels will be marked red and a new calibration phase needs to be run after having improved the contact between optodes and skin. Important is that the displayed layout is qualitatively reproducible across different subjects attending the experiment.

Signal level: Signal levels and noise ratios can be examined for each wavelength differently to the gain levels. Signal levels are mapped according to a logarithmic scale and expressed in voltage units.

Noise: In order to quantify the noise level, the system uses coefficient of variation (CV = standard deviation/mean × 100) of those recorded data used to assess signal levels.

Dark noise: high levels of dark noise may be due to environmental light disturbance or if the NIRS system is connected to power.

As default, the color combination is set according with the traffic light colors (excellent, acceptable, critical and lost). If "Quality Scale" button is deactivated, the older color scheme is displayed as shown in **Figure 4**.

#### **2.2. Data visualization and recording**

If the configuration phases and the calibration have been followed correctly, real-time data are displayed in a preview or can be directly recorded. Side-by-side there is a trace display and a topographic map for Hb and HbO concentration (**Figure 5**). Data presented in the topographic map represent estimated changes of oxy and deoxyhemoglobin concentration. The recorded data are raw signals of individual wavelengths.

The estimated data of oxy and deoxyhemoglobin concentration vary for each channel and are shown on the left side of the data visualization panel. In order to set an appropriate rehabilitation approach this allows us to visually inspect the ongoing fluctuations and verify them according to a given stimulus. For a clinical assessment, being able to analyze temporal sequences of each channel by selecting them individually is important. Amplifying the signal level (**Figure 6**) allows us to enlarge the fluctuations in order to better inspect the recording data. If no scale factor is applied, the unit of Hb state is mmol/L but may be scaled up to a factor of 2000 mmol/L.

Before starting the recording, in the preview data visualization, it is important to apply the low-pass frequency filter to remove high-frequency noise or the heartbeat component (**Figure 7**).

Despite the trace display, the Hb-HbO concentration is shown as a 2D map where channels are depicted (**Figure 8**).

In order to enhance Hb dynamics on the display, the Hb gain can be scaled up for better clinical observations purposes. The trace display in **Figure 8** has been filtered to remove high

**Figure 4.** (Courtesy of NIRx Medical Technologies) shows gain map, signal level and noise in a topographic layout for each channel. In the left column, the option "quality scale" is activated; in the right column, the option "quality scale" is

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frequency and 1 Hz heartbeat frequency.

deactivated, where the only thing changing is the color scheme.

By clicking on the "Details" button, a detailed calibration window will open with individual maps of gain, level and noise. Gain map: an inverse relationship exists between gain and intensity of the received light, intensity of light must be more amplified if the light signal is weak. If the outdistance between sources and detectors is between 2.5 and 3.0 cm, we expect a gain of 4, if lower than 4 there is a low level of light attenuation. If channels do not reach a maximum gain level of 7, the signal levels will be marked red and a new calibration phase needs to be run after having improved the contact between optodes and skin. Important is that the displayed

layout is qualitatively reproducible across different subjects attending the experiment.

dard deviation/mean × 100) of those recorded data used to assess signal levels.

voltage units.

90 Prefrontal Cortex

NIRS system is connected to power.

is displayed as shown in **Figure 4**.

factor of 2000 mmol/L.

are depicted (**Figure 8**).

(**Figure 7**).

**2.2. Data visualization and recording**

recorded data are raw signals of individual wavelengths.

Signal level: Signal levels and noise ratios can be examined for each wavelength differently to the gain levels. Signal levels are mapped according to a logarithmic scale and expressed in

Noise: In order to quantify the noise level, the system uses coefficient of variation (CV = stan-

Dark noise: high levels of dark noise may be due to environmental light disturbance or if the

As default, the color combination is set according with the traffic light colors (excellent, acceptable, critical and lost). If "Quality Scale" button is deactivated, the older color scheme

If the configuration phases and the calibration have been followed correctly, real-time data are displayed in a preview or can be directly recorded. Side-by-side there is a trace display and a topographic map for Hb and HbO concentration (**Figure 5**). Data presented in the topographic map represent estimated changes of oxy and deoxyhemoglobin concentration. The

The estimated data of oxy and deoxyhemoglobin concentration vary for each channel and are shown on the left side of the data visualization panel. In order to set an appropriate rehabilitation approach this allows us to visually inspect the ongoing fluctuations and verify them according to a given stimulus. For a clinical assessment, being able to analyze temporal sequences of each channel by selecting them individually is important. Amplifying the signal level (**Figure 6**) allows us to enlarge the fluctuations in order to better inspect the recording data. If no scale factor is applied, the unit of Hb state is mmol/L but may be scaled up to a

Before starting the recording, in the preview data visualization, it is important to apply the low-pass frequency filter to remove high-frequency noise or the heartbeat component

Despite the trace display, the Hb-HbO concentration is shown as a 2D map where channels

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**Figure 4.** (Courtesy of NIRx Medical Technologies) shows gain map, signal level and noise in a topographic layout for each channel. In the left column, the option "quality scale" is activated; in the right column, the option "quality scale" is deactivated, where the only thing changing is the color scheme.

In order to enhance Hb dynamics on the display, the Hb gain can be scaled up for better clinical observations purposes. The trace display in **Figure 8** has been filtered to remove high frequency and 1 Hz heartbeat frequency.

In clinical assessment, responses to psycho-physiological stimuli (audio, visual, tactile, etc.) are interesting to examine; therefore, NIRS signal must be correlated to event-related stimuli. Markers are necessary to set the beginning and the end of a task or subjects response. NIRS can be provided with internal or external trigger signals (**Figure 9**) coming from other equip-

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The software allows to set markers manually during the experiment in case of unexpected

A block average feature allows to visualize real-time topographic areas in multiple conditions

**Figure 8.** (Courtesy of NIRx Medical Technologies) shows the data visualization panel. On the right side of the screen, Hb and HbO concentration are represented for each channel, and on the topographic map, there are channel numbers

**Figure 9.** (Courtesy of NIRx Medical Technologies) shows different trigger signals that can be used to set the end and the begging of psycho-physiological stimuli; the marker is set by clicking on the respective button (e.g., F1) or by pushing

ment that allows stimulus presentations.

(**Figure 10**).

or source-detector labels.

F1 key on the computer keyboard.

experimental event such as motion artifacts or subject distraction.

**Figure 5.** (Courtesy of NIRx Medical Technologies) Data visualization with a trace display on the left side of the screen, topographic layout on the right side of the screen expressing changes in Hb-HbO concentration. Remember that the recorded data are raw data, based on individual wavelengths.

**Figure 6.** (Courtesy of NIRx Medical Technologies) This scale factor allows signal levels to be increased from 1 to 2000 mmol/L.

**Figure 7.** (Courtesy of NIRx Medical Technologies) shows the low-pass filter; in this case, the 1 Hz heartbeat frequency is filtered out.

In clinical assessment, responses to psycho-physiological stimuli (audio, visual, tactile, etc.) are interesting to examine; therefore, NIRS signal must be correlated to event-related stimuli. Markers are necessary to set the beginning and the end of a task or subjects response. NIRS can be provided with internal or external trigger signals (**Figure 9**) coming from other equipment that allows stimulus presentations.

The software allows to set markers manually during the experiment in case of unexpected experimental event such as motion artifacts or subject distraction.

A block average feature allows to visualize real-time topographic areas in multiple conditions (**Figure 10**).

**Figure 8.** (Courtesy of NIRx Medical Technologies) shows the data visualization panel. On the right side of the screen, Hb and HbO concentration are represented for each channel, and on the topographic map, there are channel numbers or source-detector labels.

**Figure 9.** (Courtesy of NIRx Medical Technologies) shows different trigger signals that can be used to set the end and the begging of psycho-physiological stimuli; the marker is set by clicking on the respective button (e.g., F1) or by pushing F1 key on the computer keyboard.

**Figure 7.** (Courtesy of NIRx Medical Technologies) shows the low-pass filter; in this case, the 1 Hz heartbeat frequency

**Figure 5.** (Courtesy of NIRx Medical Technologies) Data visualization with a trace display on the left side of the screen, topographic layout on the right side of the screen expressing changes in Hb-HbO concentration. Remember that the

**Figure 6.** (Courtesy of NIRx Medical Technologies) This scale factor allows signal levels to be increased from 1 to 2000

recorded data are raw data, based on individual wavelengths.

is filtered out.

mmol/L.

92 Prefrontal Cortex

### **2.3. Topographic display**

By using a NIRS technique, a rendering function is useful since it provides a hemoglobin topographic data into realistic 2D-3D coordinates. This function is given by another software that is in any case necessary in order to visualize hemoglobin fluctuations following psychophysiological stimuli (**Figure 11**).

Once the software is configured, the cap can be placed on the subject's head according to the

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Before starting to record, a calibration phase is necessary. The system will automatically determine the quality of emitted (sources) and detected (detectors) light signals by assigning a quality indicator for each defined channel. If optodes are placed correctly, signal quality is good or acceptable (see **Figure 3**) and the subject is in resting state, the calibration session

Signal quality derives from multiple factors such as photodetectors' amplification level, estimated noise level (carefully inspect environmental light interferences), optode and skin contact, optimal distance between sources and detectors (30 mm). At the end of calibration, the channel quality signal allows to identify if the following steps have been considered:

**5.** Avoid spreading of environmental light into the cap; do not place the subject under a bright light or put on the cap an additional black cap that avoids light to pass through. **6.** Optode perpendicularity (both sources and detectors must stay in vertical position

**7.** Skin color, hair color or hair products such as hair gel can influence light reflection and

The criterion is to adjust signal quality each time in order to achieve a channel quality that is colored in green or yellow, not red or white that describes a critical loss of signal and a

If everything is fine, each channel is in its optimal condition and the recording phase can take

The main challenge for researchers is to apply NIRS technology to emotional research as

The first problem is represented by noise, caused by heart-rate variation and Peripheral responses following emotional stimulation. Physical changes often go along with induced state of arousal such as facial muscle contraction or, as said before, increase in heartbeat. The NIRS technique can mitigate this problem by downranging the heartbeat frequency rate (see

Section 2.2) although, if not properly set, this can bring to error of data assessment.

**2.** Check sources and detectors position according to the defined montage.

**4.** In case of complete loss of all channels, check the cable connection.

consequent exclusion of that channel during the subsequent data analysis.

**3.** Optimal distance between sources and detectors: 30 mm.

pre-selected montage, in this case the prefrontal cortex montage.

can start.

**1.** Optode to skin optical contact.

attached to the subject's skin).

**3. fNIRS: Beware of methodology!**

standardized NIRS and fNIRS methods are not yet available.

absorption.

place.

#### **2.4. How to set and prepare the NIRS system properly: a recap**

The NIRS hardware is attached to a pre-configured tablet or PC throughout a USB 2.0 cable. Every NIRS montage cap, on which sources and detectors are attached, follows the 128 standard EEG positions (known as the 10/20 international system).

**Figure 10.** (Courtesy of NIRx Medical Technologies) shows an example of block averaging display that has to be set before starting the recording session. Number of conditions need to be arranged in order to identify them graphically. The oxy (red) and deoxyhemoglobin (blue) traces displayed are a mean of the fluctuation during a fixed stimulus duration (3–10 s). Stimulus duration and number of conditions are required to be set before the recording session.

**Figure 11.** An example of the possible topographic view, in 2D or 3D mapping. (Courtesy of NIRx Medical Technologies).

Once the software is configured, the cap can be placed on the subject's head according to the pre-selected montage, in this case the prefrontal cortex montage.

Before starting to record, a calibration phase is necessary. The system will automatically determine the quality of emitted (sources) and detected (detectors) light signals by assigning a quality indicator for each defined channel. If optodes are placed correctly, signal quality is good or acceptable (see **Figure 3**) and the subject is in resting state, the calibration session can start.

Signal quality derives from multiple factors such as photodetectors' amplification level, estimated noise level (carefully inspect environmental light interferences), optode and skin contact, optimal distance between sources and detectors (30 mm). At the end of calibration, the channel quality signal allows to identify if the following steps have been considered:

**1.** Optode to skin optical contact.

**2.3. Topographic display**

94 Prefrontal Cortex

physiological stimuli (**Figure 11**).

**2.4. How to set and prepare the NIRS system properly: a recap**

dard EEG positions (known as the 10/20 international system).

By using a NIRS technique, a rendering function is useful since it provides a hemoglobin topographic data into realistic 2D-3D coordinates. This function is given by another software that is in any case necessary in order to visualize hemoglobin fluctuations following psycho-

The NIRS hardware is attached to a pre-configured tablet or PC throughout a USB 2.0 cable. Every NIRS montage cap, on which sources and detectors are attached, follows the 128 stan-

**Figure 11.** An example of the possible topographic view, in 2D or 3D mapping. (Courtesy of NIRx Medical Technologies).

**Figure 10.** (Courtesy of NIRx Medical Technologies) shows an example of block averaging display that has to be set before starting the recording session. Number of conditions need to be arranged in order to identify them graphically. The oxy (red) and deoxyhemoglobin (blue) traces displayed are a mean of the fluctuation during a fixed stimulus duration (3–10 s). Stimulus duration and number of conditions are required to be set before the recording session.


The criterion is to adjust signal quality each time in order to achieve a channel quality that is colored in green or yellow, not red or white that describes a critical loss of signal and a consequent exclusion of that channel during the subsequent data analysis.

If everything is fine, each channel is in its optimal condition and the recording phase can take place.
