Reliable Acquisition of Electroencephalography Data during Simultaneous Electroencephalography and Functional MRI

The research ethics committee of Osaka University Hospital and the safety committee of the Center for Information and Neural Networks (CiNET) approved the protocol (Osaka University Hospital Approval Nos. 18265 and 19259; CiNET Approval Nos. 2002210020 and 2002120020). All subjects provided written informed consent for their participation.

1. Preparation of the experimental setup

1. Connect the MRI-compatible EEG and bipolar amplifiers to the battery packs (ensure that they are fully charged) and to the recording computer.
2. Ensure that the workspace of the recording software is correctly set up. Set the amplitude resolution to 0.5 µV to avoid amplifier saturation; set the frequency filters according to the frequency band of interest. Set the sampling rate at 5,000 Hz (maximum possible for the amplifiers used in this protocol), regardless of the frequency band of interest.
   NOTE: Amplitude resolution at 0.5 µV corresponds to a maximum value of 16.38 mV, which is sufficient to record the gradient artifact, considering that gradient artifact peaks can reach amplitudes over 100-fold more than those of spontaneous scalp EEG (approximately 10-100 µV) at high speeds (>1,000-fold faster than the rate of change of ongoing EEG). Theoretically, the sampling rate should be at least twice as high (Nyquist theorem) as the highest frequency in the gradient switching spectrum, in order to accurately sample the high-frequency gradient-switching artifacts and detect the true onset of each volume's gradient activity for subsequent removal^12,18. However, increasing the sample rate results in large file sizes, which require significant investment for data storage and may also impede subsequent post-processing. Using the syncing device makes it unnecessary to raise the sample rate to improve synchronization between the EEG and MR clocks (see step 1.4). A sampling rate of 5,000 Hz is adequate for usual EEG/event-related potential (ERP) recordings, and higher sample rates do not improve data quality because the subsequent artifact correction process, which involves down sampling of the data to a frequency below 500 Hz and additional low-pass filtering, eliminates all high-frequency gradient correction residuals that may exist¹⁸.
3. Refer to the manual for details regarding the proper settings of the recording software needed for EEG acquisition in the MRI, which differs from that outside the MRI.
4. Check whether markers from the scanner, i.e., the markers for clock synchronization (sync on by default) and volume trigger (R128 by default), are periodically displayed in the online EEG recording. The sync on display indicates that the MRI scanner and EEG clocks are synchronized, and R128 indicates that the volume triggers are recorded for subsequent post-processing. The MRI scanner and EEG clocks are synchronized using the SyncBox device, which detects the scanner clock output (usually 10 MHz and above), downsamples, and outputs the clock signal (and the synchronization markers) to the USB2 interface.
   NOTE: The USB2 interface sends the EEG data from all of the amplifiers, which are phase locked to the scanner clock signal, to the recording computer¹⁸. Periodic sync on markers are triggers generated from the scanner electrical pulse to synchronize the EEG signal sampling by the MR scanner rate, a requisite for scanner artifact correction. Volume triggers are used to identify MR volume scan onset time for scanner artifact correction during offline EEG processing¹⁹.
5. Set up the MRI scanner according to the need and availability. It is best to use a transmit and receive head radio frequency (RF)-coil to minimize the risk of RF heating. However, a whole body transmit RF-coil and a 20-channel head receive only RF-coil were used here because a transmit and receive head coil was not available for the scanner used (typically the case for most modern scanners).
6. Load a 10 mL syringe (or several as needed) with the abrasive conductive gel for application of the EEG cap. One could preload the abrasive gel in a 50 mL large-capacity plastic syringe for fluid dispensing and fill the 10 mL syringe with the gel prior to the subject's arrival.
   ​NOTE: Application of a 32-channel EEG cap typically consumes about 20-25 mL of gel.

2. Applying the EEG cap and ECG electrode

1. At recruitment, ask the subject to complete a checklist of potential contraindications for MRI. Confirm that the subject has no contraindications for MRI prior to arrival.
   NOTE: In general, any subject who qualifies for MRI can participate in an EEG-fMRI study. The exclusion criteria are: non-cooperative or non-compliant subjects; those with underlying conditions (e.g., chronic back pain), which prevent them from lying supine for a certain period of time (typically at least 1 h); or subjects who may be unable to lie still on the MRI table during the scan. Movement not only hampers the quality of both EEG and fMRI data but also imposes a potential hazard to the subjects themselves (e.g., induces current in the wires and cables that may cause stimulation). In the case of task-based acquisition, the language comprehension ability of the subject should also be considered (avoid subjects who are unable to understand the instructions). In this study, 32 healthy volunteers (mean age, 40 years; 17 women) and 25 patients with epilepsy (mean age, 31 years; 13 women) were recruited.
2. Ask the subjects to wash their hair with shampoo without conditioner or wax before arrival.
3. Explain the purpose of the experiment and next steps to the subject.
4. Measure the head circumference (i.e., occipital frontal circumference) by wrapping a flexible non-stretchable measuring tape around the head over the supraorbital ridges and the occiput and select an appropriately sized cap. Use a cap that is 1 cm larger than the head circumference, and always ask the subject whether the cap is comfortable once placed (i.e., not too tight).
5. After placing the cap at the approximate position over the subject's head, using the same measuring tape, measure the lengths of the inion-nasion arc, defined as the arc over the midline of the head extending from the occiput to the bridge of the nose, and the peri-auricular arc, defined as the arc extending between the ears that crosses the midpoint of inion-nasion arc, over the cap. Mark the intersection of the inion-nasion arc and the peri-auricular arc (the point where the midpoints of both arcs meet, AKA Cz), and slide the cap over the head so that the position of electrode Cz is adjusted to this intersection. Ensure that the cap is not rotated horizontally by manually checking whether electrodes Fz, Pz, Oz, Reference, and Ground are positioned over the inion-nasion arc.
6. Expose the skin underneath each electrode by displacing the hair to the side of the electrode using the back of a cotton swab.
7. Rub the skin beneath each electrode by quickly spinning a cotton swab containing 70% alcohol solution placed through the opening of the electrode.
8. Apply a small amount of the abrasive conductive gel (~0.2 mL) in the opening and abrade the skin by quickly spinning a cotton swab in a similar fashion.
9. Monitor the impedance of the electrode (displayed by the recording software) and repeat the abrasion as stated in step 2.8 until the impedance drops at least below 20 kΩ²⁰, preferably as low as possible (below 5 kΩ)²¹.
10. Fill the opening with the same gel (usually ~0.5 mL) once the impedance is satisfactory. Do not apply excessive gel in the opening to avoid bridging between electrodes. Move to the next electrode if the impedance is unsatisfactory despite repeated abrasion and come back later because sometimes the impedance continues to drop with time after applying the gel.
11. Repeat steps 2.6-2.9 for all of the scalp EEG electrodes.
12. Before placing the ECG electrode at the back, ask the subject to sit upright without flexing the neck.
13. Make sure that the ECG electrode wire is straight when placing the ECG electrode at the back but keep some allowance for laying the ECG electrode wire along the curve of the neck, to avoid displacement of the electrode when the subject lays down on the MRI table. Place the ECG electrode 2-3 cm left from the median furrow, which can be identified as the vertical indentation along the midline of the back. The vertical position varies depending on the subject's height; it is typically positioned on the lower back approximately on the line that extends between the tips of the scapula in a subject of about 160 cm.
14. Rub the skin underneath the ECG electrode with an alcohol swab.
15. Attach the ECG electrode to the skin using a double-sided adhesive ring and repeat steps 2.8-2.9. The adhesive ring also serves as a padding to avoid direct contact of the electrode with the skin.
16. Fold the dry alcohol cotton swab into four, and place it on the ECG electrode. Tape it to the skin using a surgical tape (medical adhesive tape). Tape the ECG electrode wire to the skin up to the shoulder.

3. Apply the carbon wire loop (if a bipolar amplifier is available)

1. Place a set of pre-braid carbon wire (diameter 1 mm)⁹ consisting of six loops (diameter 10 cm) over the cap in a position such that the bundle of the wires come in parallel with the bundle of the electrodes on the top of the head.
2. Use surgical tape (1 x 2 cm) to secure the loops around the electrodes, so that the loops cover the head with each loop covering evenly almost an equal area (i.e., both the fronto-temporal, both the temporo-occipital, the occipital, and the vertex). Alternatively, one could also sew the loops to the EEG-cap, if applicable.
   ​NOTE: The carbon wire loops on the head serve to capture movement, including ballistocardiogram (BCG). These signals are used for the removal of BCG artifacts from the EEG during offline EEG processing⁹.

4. Securing the cap and carbon wire loops

1. Make sure that the EEG electrodes are not forming loops.
2. Wrap the subject's head with an elastic bandage over the EEG cap and the carbon loops. The bandage serves to press the EEG electrode tightly onto the skin, to reduce MRI machinery-induced vibration of the electrodes and prevent the gel from spilling onto the pillow when placing the subject inside the MR scanner (see step 5).
3. Make sure that the bandage covers all of the electrodes and is not too tight by asking whether the subject feels uncomfortable pressure on the head while applying the bandage.

5. Placing the subject in the MR scanner

1. In the case of resting-state acquisition, instruct the subject to apply MRI-compatible earbuds in the ears. In the case of task-based acquisition, instruct the subject to apply the MRI-compatible headset or earphones as per the requirement of the experiment. Make sure that the subject can hear through both sides of the headset or earphones.
2. Place an MRI-compatible flat memory foam pillow in the lower half of the head coil before asking the subject to lie down and place the head in the coil.
3. After positioning the head appropriately (top of the head placed as close as possible to the top of the head coil), place the electrode and carbon wire bundles straight through the top opening of the head coil.
4. Add memory foam pillows to the top of the head, forehead, and temporal area. The pillows should appropriately fill up all spaces left within the head coil while not compressing the subject's head too tightly.
   1. Make sure that the pillows are not squeezing the head while placing the upper half of the head coil and while closing the coil. Adjust the pillows or change to smaller size pillows if too tight. In this way, the pillows serve to hold the electrode wires to reduce MRI machinery-induced vibration on the electrode wires and to restrain head movements while maintaining the comfort of the subject during the scan.
   2. Place a half cylinder-shape memory foam pillow at the back of the neck so that the ECG electrode wire is sandwiched well between the pillow and the neck. The portion of ECG electrode wire that passes at the back below the shoulder is indeed sandwiched between the back of subject and the MRI table and is thus immobilized by the subject's own weight.
5. In the case of a task-based acquisition, after placing all of the memory foam pillows, make sure that the headset or earphones are not displaced by testing again if the subject can still hear through both sides of the headset or earphones. After closing the head coil, place the mirror and instruct the subject to adjust the mirror (in the case of the task that requires visual stimuli). Instruct the subject to adjust the mirror if necessary, after moving the table in order to place the subject's head at the isocenter of the MRI bore.
6. Connect the amplifiers placed at the back of the MRI bore to the recording computer placed in the console room using the optic fibers provided.
7. After connecting the EEG/ECG electrodes and the carbon wire loops to the EEG and the bipolar amplifiers at the back of the MRI bore, switch on the amplifiers. Again, check the impedance of all electrodes to make sure that they are still low (at least below 20 kΩ). Remove the subject from the MR scanner for adjustment if there is any electrode with high impedance.

6. Configuration of the wires and amplifiers

1. Arrange all of the wires between the outlet of the top opening of the head coil and the amplifiers (including the electrodes and carbon wire bundles, the connector box, and the ribbon wires) so that they are placed straight and at the center of the MRI bore. This is important to minimize MRI-induced current.
2. Place one carbon wire loop around the ribbon cable going from the EEG/ECG electrodes connector box to the amplifier and connect all of the carbon wire loops (see step 5.7) to the input box of the bipolar amplifier (EXG MR). This loop serves mainly to capture the vibrations caused by the Helium pump⁹.
3. To minimize MRI machinery-induced vibration, immobilize the wires by sandwiching all of them with MR-safe and non-ferromagnetic sandbags all along the way between the outlet of the top opening of the head coil and the amplifiers. Also, place sandbags on the amplifiers. These sandbags, measured 330 mm x 240 mm x 50 mm and weighing 4 kg, are supplied by the EEG manufacturer.
4. Position the amplifiers outside the bore of the magnet, which is allowed by the length of the cables as supplied by the manufacturer.

7. EEG-fMRI data acquisition

1. Make sure that the subject is comfortable with the positioning before leaving the scanner room, to avoid unnecessary subject movement during the acquisition. Instruct the subject to press the alarm button if necessary (i.e., in case of emergency or if the subject feels an uncomfortable sensation). Communicate with the subject from the console room to confirm that the subject can hear the operator. Tell the subject that loud noises are expected during data acquisition. Instruct the subject as required for the experiment, and instruct the subject not to move during data acquisition.
2. Start the EEG recording before starting fMRI acquisition. Typically, the following images are acquired sequentially: scout images (two-dimensional) for positioning the fMRI field of view, fMRI, and structural images for co-registering the fMRI images during postprocessing. Shim sequences were run prior to acquiring each type of image for calibration of appropriate parameters.
   NOTE: It is important to use MRI sequences that are proven safe with amplifiers to maintain safety, and to avoid any damage to the amplifiers¹⁸. Details regarding the sequences considered safe will not be discussed in detail. Readers are encouraged to consult the user manual or the support team. In general, gradient echo sequences are recommended and spin echo sequences or any sequence with equivalent RF emission parameters, which can cause excessive RF-induced heating, should be avoided. Heating can be indirectly quantified using metrics that measure the amount of RF exposure, such as specific energy absorption rate (SAR) and the root mean square value of B₁+ averaged over 10 s (B₁+rms). Recently, B₁+rms, dependent on the imaging parameters but independent of the subjects' body mass²², is becoming the new standard to specify the limit. For example, the B₁+rms thresholds for acquisition at 3 T using the Brain Products EEG cap are 1 µT for the current standard cap and 1.5 µT for the new standard EEG cap with a shorter (10 cm) bundled cable²³. Flip angle, number of slices, and repetition time (TR) are parameters that need to be considered to keep SAR and B₁+rms low. A small flip angle (<90°) is recommended. The number of slices and TR can be adjusted as long as the resulting sequence is below the threshold of B₁+rms²³.
3. Upon starting the acquisition, make sure again markers from the scanner (see 1.4) are periodically displayed in the online EEG recording.