Choosing your reference for an EEG recording and the advantage of use Analyzer.

01/12/2023 04:18:51 Author: Brain Products @edited by Noronha

Choosing your reference for an EEG recording and the advantage of use Analyzer.

EEG recordings measure differences in electrical potentials between two points (Voltages) and are usually expressed in units of microvolts (µV). This means the signal displayed at any channel is in fact the difference in electrical potential to some other recording site. Primarily, this other recording site is the ground electrode. As the name indicates, this electrode is connected to the ground circuit of the amplifier, and it thereby picks up electrical noise that does not reach the other scalp electrodes. Consequently, the voltage difference between ground and EEG scalp electrodes is also affected by this noise.

This ground electrode is supposed to be acquired from the same region for all the channels (for example Cz) and can be acquire for 2 regions (for example M1 + M2). Check the image below for some examples of reference channels: (YAO et al. 2019)

There are a lot of options for references. On the blog we are going to analyze how the reference affect the data, how can we change the references after collect the data and what happens during re-referencing. This blog provides some useful information about referencing that might help you choosing the optimal reference for your data analysis.

1 Which online reference should I use?

"The ideal reference should be the one with zero or constant potential but unfortunately it is well known that no point on the body fulfills this condition. Consequently, more than ten references are used in the present EEG-ERP studies. This diversity seriously undermines the reproducibility and comparability of results across laboratories." (Yao D. et al; 2019)

Many EEG caps have a pre-defined reference channel, for example at the position of FCz or Cz. In other cases, dedicated electrodes can be attached to the nose, earlobes or mastoids (the bones behind the ears) to serve as online reference. In general, it is recommended to use a single channel as online reference during recording (instead of e.g. physically linked mastoids). Otherwise neither the contributions nor the signal quality (impedances) of the different channels forming the online reference can be disentangled and controlled.

You will find that the reference itself is not displayed as a channel in your data. Recording the reference against itself (online reference – online reference) would mean amplifying and recording zero Volt differences. The online reference is hence not available as a channel with meaningful information.

However, note that the choice of the online reference is not too decisive: you still have the option to choose a different reference during offline referencing

2 Re-referencing after recording – the offline reference

Changing the reference offline after recording is called re-referencing. The idea behind re-referencing is to express the voltage at the EEG scalp channels with respect to another, new reference. It can be composed of any recorded channel or an average of several channels. This ultimate reference for your data will also affect your analysis and should be chosen with care.

2.1 How does re-referencing work?

Before re-referencing, the new reference itself reflects the difference in electrical potential to the online reference (new reference – online reference). During re-referencing, the signal of the new reference is subtracted from each EEG channel. Afterwards, the voltage at these channels reflects the difference to the new reference and no longer to the online reference ([channel – online reference] – [new reference – online reference] = channel – new reference). In the same way, the new reference is subtracted from itself during re-referencing and thus reflects the new level of zero voltage.

The same process can in fact also be applied to the online reference, which – after the subtraction of the new reference – is simply the equivalent of the negated new reference ([online reference – online reference] – [new reference – online reference] = online reference – new reference). This way, the signal of the online reference, which has been implicitly contained in all other channels, can be regained with respect to a new reference. This can be useful if the online reference channel (for example Cz or FCz) is of relevance for your data analysis.

3 How does the choice of the reference affect the data?

The reference influences the amplitude at each EEG channel and time point: it defines the level of zero voltage and all other channels are expressed in relation to it. Channels that are located closely to the reference are thereby likely to be affected by similar electrical activity. Their difference in potential to the reference, i.e. their amplitude, will therefore be rather small. By contrast, channels that are distant from the reference are potentially also affected by other electrical sources (e.g. other brain activity or noise). They are more likely to display larger voltage differences to the distant reference, and thereby exhibit a larger amplitude. This way, the spatial distance to the reference can affect the amplitude of a channel, and potentially also the level of noise remaining in the signal after re-referencing. Alike the amplitude, also the polarity of the signal at different channels depends on their relationship to the reference, i.e. if their electrical potential is lower or higher in comparison. For these reasons, the offline reference is usually chosen to be symmetric. Otherwise, the voltage distribution on the head will likely be biased towards one hemisphere.

The topography of the EEG signal is shaped by the relative differences in voltage between the scalp channels. Re-referencing does not affect this distribution because the new reference is subtracted equally from all channels. However, given that the new reference determines the level of zero voltage, re-referencing can shift the overall amplitude of the topography to a higher or lower level.

4 What are commonly used referencing schemes?

The optimal reference always depends on the signal of interest. However, two commonly employed referencing schemes will be described in some more detail.

4.1 Average mastoids

The average of the two mastoids is often used as offline reference, especially if the signal in central areas or along the midline is of interest. The electrodes on the mastoids are expected to pick up relatively little cortical activity from the top of the head. At the same time, they are located closely enough to the other scalp channels to pick up similar noise. Averaging across the left and right mastoid furthermore offers a symmetric reference that should not prefer one of the hemispheres. Dedicated mastoid electrodes can be placed behind the ears (often called M1 and M2 or A1 and A2) or the mastoids can be approximated by the electrode positions TP9 and TP10.

4.2 Average of all scalp channels (Common Average Reference)

When applying the so-called Common Average Reference, the new reference is the average electrical activity measured across all scalp channels. Re-referencing is achieved by creating an average of all scalp channels and subtracting the resulting signal from each channel. After re-referencing, the overall electrical activity (amplitude) across all channels will sum up to zero at each time point. When using this reference, amplitudes will be overall reduced, but each channel will contribute equally to the new reference.

Theoretically, the re-referenced signal should not be biased towards any specific location on the scalp. However, the head is normally not evenly covered by electrodes. Instead, electrodes are often placed more densely on the top of the head and do not cover the underside at all. If the signal on top of the head is overrepresented in the reference, the amplitudes in this region will be reduced. To minimize this bias, the average reference hence requires high coverage with at least 64 to 128 electrodes (Nunez, 2006). Optimally, electrodes should thereby be evenly spaced and cover over 50% of the head surface (Luck, 2014). Luck (2014) furthermore urges reporting of exactly which channels were included in the average reference to allow replicability and comparability to other studies.

5 The New Reference transformation

In BrainVision Analyzer 2, re-referencing can be achieved by applying the New Reference transformation. It can be found under Transformations > Dataset Preprocessing > Channel Preprocessing > New Reference in Analyzer 2.1.x or under Transformations > Dataset Preprocessing > New Reference in Analyzer 2.2.x. With this transformation, you can re-reference your signal in three steps.

5.1 The steps of the New Reference transformation

5.1.1 First dialog window: Select the channels that form the new reference

In the first dialog you can select the channel(s) that should form the new reference. If several channels are selected, their average will constitute the new reference. If for example the mastoid channels M1 and M2 are selected, the new reference signal will constitute their averaged signal (M1 + M2) / 2.

The option ‘Include Implicit Reference…’ should only be selected if the (implicit) online reference channel is intended to be part of the new reference. One reason to use this option would be if one mastoid formed the online reference and you would like to re-reference to both. Another reason to include the online reference would be given when re-referencing to the average of all channels. Even though the implicit reference won’t contribute any (non-zero) amplitude values to the average calculation, it will still increase the channel number in its denominator.

5.1.2 Second dialog window: Select the channels to which the new reference will be applied

In the second dialog , you can select which channels the new reference shall be applied to. You will also have the option to keep channels that are not being re-referenced. Please note that usually all scalp channels should refer to the same reference. Otherwise you risk mixing different referencing schemes in transformations that include all scalp channels (such as topographic maps, ICA, connectivity calculations etc.).

5.1.3 Third dialog window: New and old reference channel

If the ‘Name of New Reference Channel’ option is selected in the third dialog , the chosen name will be added as a suffix to all re-referenced channels.

If the box ‘Reuse Old Reference Channel’ is ticked, you can reconstruct the signal of the online reference channel with respect to the new reference. The new reference is then subtracted from the online reference, which will after this step consist of the negated new reference.

6 What can I do if the Ground is with noise?

To eliminate this ground-related noise, an online reference is chosen out of the recorded EEG channels, which is used as “baseline” for all remaining EEG channels. This means the signal at the other EEG channels is expressed as the difference in electrical potential to this reference instead of to the ground. Relating the EEG channels to the reference cancels out the noise stemming from the ground circuit ([channel – ground] – [online reference – ground] = channel – online reference). Differential amplifiers amplify this (less noisy) voltage difference between EEG channels and reference. Therefore, when looking at your EEG data, keep in mind that the amplitude at any channel constitutes the difference in electrical potential to the chosen reference.

References

YAO, Dezhong et al. Which Reference Should We Use for EEG and ERP practice?. Brain topography, p. 1-20, 2019.

https://pressrelease.brainproducts.com/referencing/

Li, Y., Wang, Y., Zhang, B., Wang, Y. & Zhou, X. (2018). Electrophysiological Responses to Expectancy Violations in Semantic and Gambling Tasks – A Comparison of Different EEG Reference Approaches. Frontiers in Neuroscience, 12(169), 1-15.

Luck, S. J. (2014). An introduction to the event-related potential technique, Cambridge: MIT press.

Nunez, P. L. and R. Srinivasan (2006). Electric fields of the brain: the neurophysics of EEG. Oxford: University Press.

Yao, D. (2001). A method to standardize a reference of scalp EEG recordings to a point at infinity. Physiological Measurement, 22(4) 693-711
Dien, J. (1998). “Issues in the application of the average reference: Review, critiques, and recommendations.” Behavior Research Methods, Instruments, & Computers 30(1): 34-43.