This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.
Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.
Electroencephalography (EEG) has been widely used to record electrical activity of the human brain both in clinical diagnosis and scientific research, since it is noninvasive, low-costed and has very high temporal resolution1. In order to study the EEG signals in resting state, researchers have developed many EEG techniques (e.g., power spectrum analysis, functional connectivity analysis)2,3. Of these, microstate analysis and omega complexity analysis could make good use of the spatial and temporal information inherent in EEG signals4.
Previous researches have shown that although the topographical distribution of EEG signals varies over time in eye-closed or eye-open resting state, the series of momentary maps show discontinuous changes of landscapes, i.e., periods of stability alternating with short transition periods between certain quasi-stable EEG topographies5. Microstates are defined as these episodes with quasi-stable EEG topographies, which last between 80 and 120 ms1. Since different electric potential landscapes must have been generated by different neural sources, these microstates may qualify as the basic blocks of mentation and can be considered as "atoms of thought and emotion"6. Using modern pattern classification algorithms, four resting EEG microstate classes have been consistently observed, which were labeled as class A, class B, class C and class D7. Moreover, researchers revealed that these four microstate classes of resting EEG data were closely associated with well-known functional systems observed in many resting-state fMRI (functional magnetic resonance imaging) studies8,9.Thus, the microstate analysis provided a novel approach to study the resting state networks (RSNs) of human brain. In addition, the average duration and the frequency of occurrence of each microstate class, the topographical shape of the four microstate maps are significantly influenced by some brain disorders4,10,11, and are associated with fluid intelligence12 and personality13.
In the other aspect, traditional functional connectivity of multi-channel EEG could only describe the functional connections between two scalp electrodes, thus failed to assess the global functional connectivity across scalp or within a certain brain region. The omega complexity, proposed by Wackermann (1996)14 and calculated through an approach combining principal component analysis (PCA) and Shannon entropy, has been used to quantify the broad-band global synchronization between spatially distributed brain regions. In order to assess the omega complexity of each frequency band, Fourier transform was commonly conducted as an initial step25.
The microstates and omega complexity can be used to reflect two closely linked concepts, i.e., the temporal complexity and spatial complexity4. Since the microstate classes represent certain mental operations in human brain, they can reflect the temporal structure of neuronal oscillations. Lower duration and higher occurrence rate per second must indicate higher temporal complexity. The omega complexity is positively related with the number of independent neural sources in brain, thus are commonly considered as an indicator of spatial complexity4.
The current article describes the protocol of EEG microstate analysis and omega complexity analysis in detail. The EEG microstate and omega complexity analyses offer the opportunity to measure the temporal and spatial complexities of brain activity respectively.
This protocol was approved by the local ethical committee. All the participants and their parents signed an informed consent form for this experiment.
1. Subjects
2. EEG data recording
3. EEG Data Preprocessing
NOTE: EEG data could be preprocessed using various open source or commercial software. The instructions provided below are specific for EEGLAB. This is only one out of many available options to preprocess EEG data.
4. EEG Microstate Analysis
NOTE: A modified version of the classical K-means clustering algorithm is used for microstate class analysis16, which contains a bottom-up procedure and an up-bottom procedure. In the bottom-up procedure, the group-level microstate classes are identified using the spatial correlation as a clustering criterion. Then in the up-bottom procedure, each topographical map of each subject in each group is assigned to the EEG microstate class with maximum spatial correlation. For resting-state EEG microstate analysis, the polarity of topographical maps is commonly disregarded. EEG microstate class analysis could be done using various open source softwares, such as CARTOOL, sLORETA, EMMA and MapWin. The instructions provided below are specific for the EEGLAB plugin for Microstates. This EEGLAB plugin could be downloaded from https://sccn.ucsd.edu/wiki/EEGLAB_Extensions_and_plug-ins.
5. Omega Complexity Analysis
EEG microstate
Grand mean normalized microstate maps are displayed in Figure 1. The electric potential landscapes of these four microstate classes identified here are very similar to those found in previous studies4.
The mean and standard deviation (SD) of microstate parameters of the healthy subjects were shown in Table 1. For microstate class A, the occurrence rate was 3.44 ± 1.29 times/s, and the duration was 72 ± 13 ms. For microstate class B, the occurrence rate was 3.54 ± 0.85 times/s, and the duration was 71 ± 18 ms. For microstate class C, the occurrence rate was 3.85 ± 0.63 times/s, and the duration was 69 ± 9 ms. For microstate class D, the occurrence rate was 3.41 ± 0.78 times/s, and the duration was 66 ± 11 ms.
Omega Complexity
The value (mean ± SD) of global omega complexity of each frequency band in the healthy subjects was presented in Table 2. For delta band, the global omega complexity was 6.39 ± 1.34. For theta band, the global omega complexity was 5.46 ± 0.85. For alpha-1 band, the global omega complexity was 3.47 ± 0.8. For alpha-2 band, the global omega complexity was 3.87 ± 0.70. For beta-1 band, the global omega complexity was 5.36 ± 0.84. For beta-2 band, the global omega complexity was 6.16 ± 0.83. For gamma-1 band, the global omega complexity was 6.95 ± 1.07. For gamma-2 band, the global omega complexity was 6.88 ± 1.39.
The value (mean ± SD) of anterior omega complexity of each frequency band in the healthy subjects was shown in Table 2. For delta band, the anterior omega complexity was 4.84 ± 1.7. For theta band, the anterior omega complexity was 4.23 ± 1.48. For alpha-1 band, the anterior omega complexity was 3.44 ± 1.09. For alpha-2 band, the anterior omega complexity was 3.87 ± 0.97. For beta-1 band, the anterior omega complexity was 3.74 ± 0.81. For beta-2 band, the anterior omega complexity was 2.94 ± 0.59. For gamma-1 band, the anterior omega complexity was 1.98 ± 0.24. For gamma-2 band, the anterior omega complexity was 3.02 ± 0.59.
The value (mean ± SD) of posterior omega complexity of each frequency band in the healthy subjects was shown in Table 2. For delta band, the posterior omega complexity was 3.71 ± 1.48. For theta band, the posterior omega complexity was 2.47 ± 0.85. For alpha-1 band, the posterior omega complexity was 2.11 ± 0.9. For alpha-2 band, the posterior omega complexity was 3.16 ± 1.42. For beta-1 band, the posterior omega complexity was 4.32 ± 1.67. For beta-2 band, the posterior omega complexity was 3.84 ± 1.04. For gamma-1 band, the posterior omega complexity was 2.17 ± 0.37. For gamma-2 band, the posterior omega complexity was 2.99 ± 0.53.
Figure 1. Mean normalized topographical maps of the four microstate classes (A-D) of resting-state EEG in the healthy subjects. Microstate class A and B have a right frontal to left occipital orientation and a left frontal to right occipital orientation, respectively. Microstate class C and D have symmetric topographies, but prefrontal to occipital orientation and frontocentral to occipital orientation were observed, respectively. Please click here to view a larger version of this figure.
Microstate classes | ||||||||
A | B | C | D | |||||
Mean | SD | Mean | SD | Mean | SD | Mean | SD | |
Occurrence/s | 3.44 | 1.29 | 3.54 | 0.85 | 3.85 | 0.63 | 3.41 | 0.78 |
Duration (ms) | 72 | 13 | 71 | 18 | 69 | 9 | 66 | 11 |
Table 1. Microstate parameters of the healthy subjects (n=15). The mean and standard deviation (SD) of the occurrence rate and duration of the four microstate classes were shown in this table.
Global Omega Complexity | Anterior Omega Complexity | Posterior Omega Complexity | ||||
Frequency Band | Mean | SD | Mean | SD | Mean | SD |
Delta | 6.39 | 1.34 | 4.84 | 1.7 | 3.71 | 1.48 |
Theta | 5.46 | 0.85 | 4.23 | 1.48 | 2.47 | 0.85 |
Alpha-1 | 3.47 | 0.8 | 3.44 | 1.09 | 2.11 | 0.9 |
Alpha-2 | 3.87 | 0.7 | 3.87 | 0.97 | 3.16 | 1.42 |
Beta-1 | 5.36 | 0.84 | 3.74 | 0.81 | 4.32 | 1.67 |
Beta-2 | 6.16 | 0.83 | 2.94 | 0.59 | 3.84 | 1.04 |
Gamma-1 | 6.95 | 1.07 | 1.98 | 0.24 | 2.17 | 0.37 |
Gamma-2 | 6.88 | 1.39 | 3.02 | 0.59 | 2.99 | 0.53 |
Table 2. Global, anterior and posterior omega complexity of the healthy subjects (n=15). The mean and standard deviation (SD) of the global, anterior and posterior omega complexity for the eight frequency bands (delta, theta, alpha-1, alpha-2, beta-1, beta-2, gamma-1, gamma-2) were shown respectively in this table.
Supplemental Files. In order to run the scripts used in this manuscript, please open the scripts in the MATLAB environment, then copy all the content into the command window and press the "Enter" key. Note that, the scripts only apply to our data sets. Certain modifications are needed when the scripts are applied to other data sets. Please click here to download this file.
In this article, two kinds of EEG analytic methods (i.e., microstate analysis and omega complexity analysis), measuring the temporal complexity and spatial complexity of human brain respectively, were described in detail. There are several critical steps within the protocol that should be mentioned. Firstly, the EEG data must be cleaned before the computation of the microstate and omega complexity. Secondly, the EEG data should be remontaged against the average reference before the computation of the microstate and omega complexity. Thirdly, the continuous EEG data must be segmented into epochs before the computation of the microstate and omega complexity. The length of each epoch should be 2 s. Lastly, the software that can be used in microstate analysis include Cartool (https://sites.google.com/site/cartoolcommunity/about), sLORETA (http://www.uzh.ch/keyinst/loreta.htm), and MapWin (http://www.thomaskoenig.ch/index.php/software/mapwin). The microstate analysis was conducted by means of one plugin in the EEGLAB in this study.
Although the microstate analysis conducted here was applied to resting state EEG data, it could be easily applied to event-related potentials (ERPs), which will help us uncover more information about the time courses of diverse cognitive operations in cognitive experiments, and provide a reference-free approach to perform ERP analysis18,19. Note, for resting state EEG, the polarity of topographical maps is commonly disregarded; however, for ERPs, the polarity of topographical maps should not be disregarded. A small limitation of this EEG plugin is that it could only be used for resting state EEG. For ERPs, the software Cartool may be one of the best choices. The omega complexity value attains from 1 to N. If omega complexity computed is 1, a maximum global functional connectivity within a certain brain region is revealed; whereas if omega complexity equals N, a minimum global functional connectivity within a certain brain region is found. Thus, if we want to statistically test the omega complexity of different brain regions, the number of electrodes selected in these regions must be equal, since the number of electrodes could significantly influence the value of omega complexity estimated.
In order to study the resting EEG, researchers have developed many EEG techniques (e.g., power spectrum analysis, functional connectivity analysis)2,3. Compared to these traditional techniques, microstate analysis takes full advantage of the excellent temporal resolution of EEG technique. The four identified microstate classes were found to be correlated with four well-studied functional systems observed in many resting-state fMRI studies8,20: auditory (microstate A), visual (microstate B), partially cognitive control and partially default mode (microstate C), and dorsal attention (microstate D). Thus, the microstate analysis provided a novel approach to study the resting state networks (RSNs) of human brain. Compared to traditional EEG techniques, the omega complexity could characterize the global functional connectivity within a certain brain region4. Traditional functional connectivity could only describe the functional connectivity between two scalp electrodes.
However, the two EEG techniques also have several limitations which should be mentioned. Firstly, the existing microstate analysis is commonly performed on broad-band EEG signals, thus it does not take advantage of the rich frequency information of the EEG technique. Moreover, the functional significance of these four microstate classes and related metrics are not very clear so far. Secondly, the omega complexity can only detect linear dependences. It cannot detect the nonlinear dependences between scalp regions, which could be quantified by some traditional functional connectivity metrics (e.g., phase-locking value, mutual information and synchronization likelihood)21,22,23.
In the future, the microstate analysis should be applied with source localization techniques (e.g., sLORETA, BESA, Beamforming), which will significantly enhance the spatial resolution of EEG signals. Although the microstate analysis has been widely used in resting EEG and ERPs, only a few studies have applied this technique to the time-frequency domain. For example, Jia et al.24 proposed an approach based on topographic segmentation analysis to optimally and automatically identify detailed time-frequency features. This approach could effectively exploit the spatial information of oscillatory activities. However, these applications are far from mature. For omega complexity, a normalized omega complexity is highly needed, since the value of omega complexity estimated is dependent on the number of electrodes selected. In the future, it should be applied to the time-frequency domain.
The authors have nothing to disclose.
This article was supported by the National Natural Science Foundation of China (31671141).
ANT 20 channels EEG/ERP system | ASA-Lab, ANT B.V., Netherlands | company web address: http://www.ant-neuro.com/ |
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EEGLAB plugin for Microstates | Thomas Koenig | https://sccn.ucsd.edu/wiki/EEGLAB_Extensions_and_plug-ins | |
sLORETA | Roberto D. Pascual-Marqui | http://www.uzh.ch/keyinst/loreta.htm | |
MATLAB 2010a | The MathWorks Inc. | company web address: http://www.mathworks.com/ |
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eeglab | Swartz Center for Computational Neuroscience, University of California, San Diego | https://sccn.ucsd.edu/eeglab/index.php |