Accepted Manuscript

Identification of epileptic high frequency oscillations in the time domain by

using MEG beamformer-based virtual sensors

Nicole van Klink, Arjan Hillebrand, Maeike Zijlmans

PII: S1388-2457(15)00637-9
DOI: http://dx.doi.org/10.1016/j.clinph.2015.06.008
Reference: CLINPH 2007520

To appear in: Clinical Neurophysiology

Accepted Date: 8 June 2015

Please cite this article as: van Klink, N., Hillebrand, A., Zijlmans, M., Identification of epileptic high frequency
oscillations in the time domain by using MEG beamformer-based virtual sensors, Clinical Neurophysiology (2015),
doi: http://dx.doi.org/10.1016/j.clinph.2015.06.008

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1 Identification of epileptic high frequency oscillations in the time domain by
2 using MEG beamformer-based virtual sensors
3
4 Nicole van Klinka, Arjan Hillebrandb, Maeike Zijlmansa,c
5
a
6 Brain Center Rudolf Magnus, Department of Neurology and Neurosurgery, University Medical
7 Center Utrecht, The Netherlands
b
8 Department of Clinical Neurophysiology and Magnetoencephalography Center, VU University
9 Medical Center, Amsterdam, The Netherlands
c
10 SEIN – Stichting Epilepsy Instellingen Nederand, Heemstede, The Netherlands
11
12 Corresponding author:
13 Nicole van Klink
14 University Medical Center Utrecht
15 Department of Neurology and Neurosurgery, HP C03.131
16 Heidelberglaan 100
17 3584 CX Utrecht, The Netherlands
18 Tel.: +31 887557735
19 E-mail: n.vanklink-2@umcutrecht.nl
20
21
22
23

1
24 Highlights
25 - Epileptic HFOs can be identified in non-invasive MEG in the time domain.
26 - Beamformer-based virtual sensors increase the signal to noise ratio.
27 - Virtual sensors enable detection of HFOs that were not discernible in original data.
28
29 Abstract
30 Objective High frequency oscillations (HFOs, > 80Hz) are biomarkers for epileptogenic cortex in
31 invasive and non-invasive electroencephalography (EEG). Identification of HFOs in
32 magnetoencephalography (MEG) is hindered by noise. Computing spatial filters using beamforming
33 to reconstruct time series for selected brain regions, so-called virtual sensors (VS), can increase the
34 signal-to-noise ratio. We identified HFOs in MEG in time domain using VS.
35 Methods Fifteen minutes of MEG data were selected from 12 patients. VS were placed around the
36 epileptic spikes (affected region) and in the contralateral hemisphere. VS and physical sensors were
37 reviewed for HFOs and spikes. HFO locations were compared to spikes and other clinical parameters.
38 Results Eight patients showed 78 time points with 575 HFOs in VS, 513 were in the affected region.
39 HFOs could not be identified in physical sensors for 61 of the 78 VS time points. HFOs overlapped
40 with presumed epileptogenic areas and were also visible in unfiltered VS signals.
41 Conclusion Beamformer-based VS analysis can help to identify epileptic HFOs that are not
42 discernable in physical MEG sensors.
43 Significance This approach can be extended to enable localization of non-invasively recorded HFOs.
44 This would help surgical planning and reduce the need for invasive diagnostics.
45
46 Keywords: High frequency oscillations, magnetoencephalography, epilepsy, beamforming, virtual
47 electrodes.
48
49

2
50 Introduction
51 Brain surgery can treat epilepsy when epileptic seizures originate from one specific brain area, but
52 accurate identification of the epileptogenic brain area can be difficult (Luders et al., 2006). It has
53 been shown that high frequency oscillations (HFOs) in intracranial electroencephalography (EEG) may
54 be used as a specific biomarker for epileptogenicity (Bragin et al., 1999; Jacobs et al., 2012). HFOs are
55 defined in the time domain as at least four oscillations above baseline in the filtered signal (>80Hz)
56 (Urrestarazu et al., 2007; Worrell et al., 2012). So far, HFO analysis in intracranial EEG has largely
57 been restricted to surgical candidates with refractory focal epilepsy.
58
59 Recent studies have shown that HFOs can also be identified non-invasively in scalp EEG (Andrade-
60 Valenca et al., 2011; Melani et al., 2013; Zelmann et al., 2013). Standard scalp EEG with the 10-20
61 system has a relatively poor spatial resolution though, due to the limited number of electrodes and
62 the distortion of the EEG signals by, mainly, the skull. Magnetoencephalography (MEG) is another
63 technique that is commonly used for identification of the epileptogenic zone during presurgical
64 evaluation (Barkley and Baumgartner, 2003; Fischer et al., 2005), and has a better spatial resolution
65 than EEG (Barkley and Baumgartner, 2003). Identification of HFOs in MEG would provide a spatially
66 more accurate non-invasive biomarker for epileptogenicity than HFOs in EEG. Moreover, MEG is
67 available at a much earlier stage in the diagnosis process, and for many more patients, than
68 intracranial EEG.
69
70 It has been suggested previously that HFOs can be detected in MEG (Papadelis et al., 2009), and time
71 frequency analysis has shown that MEG can contain high frequency components related to the
72 epileptogenic zone (Miao et al., 2014; Rampp et al., 2010; Tenney et al., 2014; Xiang et al., 2009).
73 However, these studies did not identify individual HFOs in the time domain, as is typically done in
74 intracranial EEG, so we do not know if these HFOs have the same morphology or even if we look at
75 the same phenomenon (Jacobs et al., 2012; Worrell et al., 2012; Zelmann et al., 2009). Beamformer
76 techniques might be useful to distinguish HFOs from noisy background activity (Guggisberg et al.,
77 2009; Miao et al., 2014; Papadelis et al., 2009). Beamforming is nowadays one of the mainstream
78 approaches for MEG source localization (Baillet et al., 2001). A beamformer reconstructs neuronal
79 activity for a particular location in the brain as the weighed contribution from the different MEG
80 sensors. These beamformer weights act as a spatial filter, attenuating noise from distant sources
81 (Adjamian et al., 2009; Hillebrand and Barnes, 2005; Vrba and Robinson, 2001). Once the
82 beamformer weights have been determined, the time domain signals can be reconstructed for each
83 desired location within the brain, also known as virtual sensors (Hillebrand and Barnes, 2005; Vrba
3
 84 and Robinson, 2001). We know that these virtual sensors can reveal information that is not
85 discernable in the physical sensors (personal communication). In this study, we aim to identify HFOs
86 in the time domain, as is custom in EEG, but has not been shown in MEG. We use beamformer virtual
87 sensors to improve the signal to noise ratio of MEG time domain signals and in that way show the
88 presence of HFOs.
89
90 Methods
91 Patients
92 Patients with refractory epilepsy in the presurgical workup for surgery at the University Medical
93 Center Utrecht and who had an MEG registration at the VU University Medical Center Amsterdam in
94 2013 were included. Patients underwent MEG registrations to improve delineation of an unclear
95 EEG-focus. Patients without epileptic spikes in the MEG registration, according to the clinical report,
96 were excluded, because spikes were used to determine the location of virtual sensors (see below). All
97 patients or their caretakers gave written informed consent prior to the clinical MEG recording.
98
99 Resting state MEG data acquisition
100 Patients were recorded with eyes closed for approximately fifteen minutes. Other recordings
101 included motor tasks and somatosensory stimulation (e.g. (Hillebrand et al., 2013)), yet these data
102 were not used in this study. Patients were recorded in supine position in order to minimize head
103 movements (which was on average 0.36cm (range 0.01-0.80cm)). MEG data were recorded with a
104 306-channel whole-head Elekta system (Elekta Neuromag Oy, Helsinki, Finland) in a magnetically
105 shielded room (VacuumSchmelze GmbH, Hanau, Germany). The MEG system comprised 102 sensor
106 units, each consisting of two orthogonal planar gradiometers and one magnetometer. Four or five
107 head-localization coils continuously recorded the position of the head relative to the MEG sensors.
108 The data were recorded with a sample frequency of 1250 Hz, an anti-aliasing filter of 410 Hz and a
109 high-pass filter of 0.1 Hz. We spatially filtered the raw data to reduce noise by using the temporal
110 extension of signal space separation (tSSS) applied with MaxFilter software (Elekta Neuromag Oy,
111 version 2.1) (Taulu and Hari, 2009; Taulu and Simola, 2006). In summary: SSS decomposes the
112 recording into a set of spherical harmonics and divides them into brain signals and signals from
113 outside the head (noise signals). The coefficients for these harmonics were estimated on sections of
114 data using a sliding window of ten seconds. Malfunctioning channels and channels with excessive
115 noise were discarded based on both automatic and visual inspection before estimation of the SSS
116 coefficients. The temporal extension of SSS was used to discard noise that SSS failed to discard; we
117 used a subspace correlation limit of 0.9 (Hillebrand et al., 2013).
4
118
119 Anatomical MRI
120 T1-weighted structural Magnetic Resonance Images (MRI) of each patient were used. The positions
121 of the head-localization coils in the MEG and the outline of the patients scalp were digitized. Surface
122 matching software developed by one of the authors (AH) was used to co-register the MRI and MEG
123 data. This resulted in an estimated co-registration accuracy of approximately 4mm (Whalen et al.,
124 2008). A single sphere was used as volume conductor model, which fitted best to the outline of the
125 scalp, as obtained from the co-registered MRI. This model was used for the dipole fitting and
126 beamformer analysis described below.
127
128 Clinical analysis
129 All MEG registrations had already been analyzed by an experienced MEG/EEG technician and
130 evaluated by a team consisting of clinicians, MEG/EEG technicians and physicists. They had reviewed
131 the physical MEG sensors and marked epileptic spikes. The clinically important spikes were fitted
132 from half-way the flank preceding the top to the top, with a dipole fit at every sample, using a single
133 moving equivalent current dipole (using xfit, version 5.5.18, Elekta Neuromag Oy). This way we
134 included the early component of the spike, which is least affected by propagation. The best dipoles
135 had a goodness of fit of 89.6% (range 81.3-96.5%) and a confidence volume of 0.06 V/mm3 (range
136 0.0-0.2 V/mm3). The locations of the fitted dipoles were used as an estimation of the epileptogenic
137 zone, which we refer to as the ‘affected region’. The homologous region in the contralateral
138 hemisphere is referred to as the ‘mirror region’.
139 We used the results of these clinical analyses as a starting point for the research presented here.
140
141 Initial data inspection
142 We checked the quality of the MEG data using standard display settings (0-100Hz, 10 s/page) and
143 with display settings to review HFOs (80Hz high pass finite impulse response (FIR) filter, 1 s/page).
144 This initial inspection showed too much noise in the high pass filtered sensors; reliable identification
145 of HFOs was not possible (Figure 1A). We performed beamformer analysis to reduce noise (Adjamian
146 et al., 2009), and to reconstruct the time series for the ‘affected region’ and ‘mirror region’.
147
148 Beamformer analysis
149 We used a scalar beamformer implementation similar to Synthetic Aperture Magnetometry
150 (Robinson and Vrba, 1999) (beamformer software, version 2.2.10, Elekta Neuromag Oy), which uses
151 singular value decomposition to determine the optimum current orientation for each location
5
152 (Sekihara et al., 2004). The data covariance for the beamformer calculations was based on marked
153 epileptic spikes, where we supplemented the spikes as already identified for clinical evaluation with
154 spikes identified for this study. We defined epileptic spikes as short paroxysmal events, clearly
155 different from background, with a duration < 70ms. A 200ms window around each marked spike was
156 used, and those signals were band pass filtered between 80 and 600Hz. These high frequency
157 components of the epileptic spikes were used to construct the data covariance. The first ten seconds
158 of the unfiltered data were used to estimate noise covariance. The noise covariance and data
159 covariances from the active periods (on average 29 periods, range 10 – 56) in both gradiometers and
160 magnetometers were used to compute the (normalised) beamformer weights for so-called virtual
161 sensors (Hillebrand and Barnes, 2005; Hillebrand et al., 2005).
162
163 Virtual sensors
164 The obtained beamformer weights were used to reconstruct the time-series for selected locations in
165 the brain, referred to as virtual sensors (Hillebrand and Barnes, 2005; Hillebrand et al., 2005). Virtual
166 sensors were placed at the location of the epileptic spikes as obtained through fitting individual spike
167 dipoles (the affected region). In order to minimize the chance of missing a focal epileptic spike or
168 HFO (Barnes et al., 2004), a set of 35 virtual sensors was used to cover the affected region. The
169 center of the virtual sensors was placed in the approximate center of the cloud of dipoles (Figure 2).
170 Virtual sensors were placed one centimeter apart (along the axes of the co-ordinate system) and
171 filled up a star-shaped configuration of six centimeter in diameter (Figure 2). As a control, another 35
172 sensors were placed at the homologous position in the contralateral hemisphere (the mirror region).
173
174 HFO and spike marking
175 The 70 virtual sensors in each patient were analyzed in the time domain in Stellate Harmonie
176 Reviewer (Montreal, QC, Canada), after down sampling to 1024 Hz. The reviewer (NvK) was blind to
177 sensor locations. First, HFOs were marked in each virtual sensor by using a symmetrical linear phase
178 63th order high pass finite impulse response (FIR) filter with a cut off frequency of 80Hz, 0.2 nAm/mm
179 amplitude scale and 1.2 seconds per page on the screen. We refer to this first round of HFO marking
180 as ‘blind marking’, because there was no knowledge of presence of epileptic spikes. After blind
181 marking of HFOs, the epileptic spikes were marked in each sensor whilst using a fourth order 100Hz
182 low pass infinite impulse response (IIR) filter, 1 nAm/mm amplitude scale and 10 seconds per page
183 on the screen. The spikes and HFOs marked by the reviewer were checked by a second reviewer (MZ)
184 and discussed until consensus was reached. Spikes and HFOs were marked on each virtual sensor

6
185 separately, and we counted how often a spike, or HFO, was visible on multiple sensors (referred to as
186 ‘spike-times’ and ‘HFO-times’).
187
188 MEG registrations contain more high-frequency artefacts than intracranial registrations
189 (Muthukumaraswamy, 2013). The resting state condition with the patient lying down was chosen to
190 minimize muscle artefacts. Inspection of the recordings learnt that high frequency background noise
191 was continuously present to a greater or lesser extent. Additionally, sharp artefacts can produce
192 HFO-like oscillations in the 80Hz high pass filtered signal (Bénar et al., 2010). Artefacts were
193 distinguished from real HFOs by eliminating oscillations with irregular morphology (Worrell et al.,
194 2012). We took a conservative approach by reviewing all marked HFOs and spikes in standard
195 settings (0-100Hz, 10 s/page) and in HFO settings (>80Hz, 1.2 s/page), and eliminating any event
196 suspected to be an artefact (Figure 3). Figure 3 A&B show an example of high frequency noise, which
197 made detection of an HFO unreliable. Figure 3 C&D show a ‘false’ HFO, which arises after filtering of
198 a sharp transient.
199
200 We then performed a second HFO marking using a different strategy, based on the knowledge that
201 many HFOs co-occur with spikes (Andrade-Valenca et al., 2011; Jacobs et al., 2008; Wang et al.,
202 2013). We evaluated the 80Hz high pass filtered signal at the moments when spikes occurred in
203 order to identify these additional HFOs; this way we marked HFOs that did not differ enough from
204 the noisy background to be reliably marked ’blindly’, but were likely to be true HFOs (Figure 4).
205
206 Statistical analysis
207 We compared the number of blindly marked HFOs and additionally marked HFOs per sensor to the
208 number of spikes per sensor using a linear mixed model, with ‘patients’ as random factor. We
209 compared the number of HFOs and spikes marked in the affected and mirror region by using the
210 Wilcoxon Signed Rank test. Statistical analysis was performed using IBM SPSS Statistics 22 (IBM Corp.,
211 Armonk, NY, USA), a p-value < 0.05 was considered significant.
212
213 HFOs in physical sensors
214 Finally, we reviewed both the gradiometers and magnetometers covering the area with virtual
215 sensors that showed HFOs, to determine if the HFOs, in retrospect, could be identified in the physical
216 sensors. For this, we used the HFO-times identified in the virtual sensors.
217
218 Comparison of HFOs to clinical parameters
7
219 The locations of HFOs were compared to the locations appointed by the SOZ, PET, SPECT, MRI and
220 clinical outcome for each individual patient with clear HFOs, i.e. > 1 HFO-time.
221
222 Time-frequency plots
223 For illustrative purposes we also computed example time-frequency plots based on the Morse
224 wavelet transform with wavelet parameters β = 6 and γ = 40.
225
226 Results
227 Patients
228 Twenty-nine patients with epilepsy from the University Medical Center Utrecht underwent a MEG
229 registration in 2013. The MEG did not show epileptic spikes in 17 patients, which left 12 patients that
230 could be included. The group consisted of 11 children, three patients were female. The resting state
231 MEG registrations were approximately 15 minutes in length, apart from patient 7, where the
232 recording lasted only 5.7 minutes. The location of the epileptic spikes as identified during the clinical
233 analysis differed between patients (Table 1). Patient 6 showed multifocal epileptic spikes, with a left
234 frontal focus as most prominent.

235
236 HFO and spike marking
237 In the virtual sensors we were able to identify HFOs that could not be discerned in the physical
238 sensors. The HFOs in the virtual sensors were also visible in the unfiltered sensors stretched in time
239 and in the time-frequency plot (Figure 1). During blind marking of HFOs, a total of 285 HFOs were
240 identified in five patients at 28 HFO-times (Table 2). 283 HFOs were identified in the affected region.
241 In four patients the HFOs were found exclusively at the side that was assigned as affected region by
242 the clinicians. Two HFOs in patient 10 were found at the side that was considered healthy and they
243 did not co-occur with a spike. HFOs were distributed over 2 to 34 out of all 70 virtual sensors. Most of
244 the HFOs co-occurred with a spike (249; 87%) and HFOs were more likely to occur in sensors with
245 spikes (t(838.64) = 10.74, p<0.001). HFOs without co-occurring spikes were identified on the same
246 sensors as HFOs with co-occurring spikes (Figure 5).
247
248 We marked a total of 20695 epileptic spikes in all patients at 1824 spike-times. Both the affected and
249 mirror region showed spikes in all but one patient (patient 7), but spikes occurred more often in the
250 affected region (median affected region: 509, mirror region: 159, Z=-2.98, p=0.003). The spikes were
251 distributed over 29 to 70 out of all 70 sensors (Table 2). Note that more than half of all marked spikes

8
252 were found in patient 8, who showed extensive spike patterns in the affected region. When we
253 exclude patient 8 from the analysis, spikes still occurred more often in the affected region (median
254 affected region: 434, mirror region: 149, Z=-2.85, p=0.004).
255
256 When specifically looking at the spike-times, another 290 HFOs were marked in seven patients at 68
257 HFO-times. Fifty of those HFO-times were new; at the other 18 we had blindly marked as HFOs on
258 other sensors. The additional HFOs were visible in the time-frequency plot (Figure 4). The additional
259 HFOs were more likely to occur in sensors with many spikes (t(839.92) = 8.27, p<0.001). Additionally
260 marked HFOs were found in three patients for whom HFOs were indiscernible during the first blind
261 marking of HFOs. Additionally marked HFOs were marked in the affected region more often than in
262 the mirror region (median affected region: 3, mirror region: 0, Z=-2.37, p=0.018).
263
264 HFOs in physical sensors
265 A total of 78 HFO-times were identified in the virtual sensors. A median of 5 channels showed an HFO
266 at an HFO time (range: 1 – 33). Using these times when inspecting the physical sensors, 61 (78%) of
267 HFO-times showed no HFO-like event (Table 2; Figure 1). HFOs were recognized in three patients in
268 physical sensors.
269
270 Comparison to SOZ, PET, SPECT, MRI and surgical outcome
271 Five patients showed clear HFOs; HFOs at more than one HFO-time. The SOZ was known in ten
272 patients and in four out of five with clear HFOs (table 1). The location of SOZ and HFOs overlapped in
273 all four. Interictal PET and/or ictal SPECT was available for nine patients, four of whom with clear
274 HFOs and in all four the location overlapped. MRI showed potential relevant abnormalities in eight
275 patients, three of whom had clear HFOs. In all three the locations of HFOs and MRI lesions
276 overlapped.
277 Six of the twelve patients had brain surgery and five patients had Engel 1 outcome. Only two (patient
278 2 and 3) out of the five patients with clear HFOs underwent surgery. The MEG affected region that
279 showed HFOs and the resected area overlapped in both patients and both were seizure free. In
280 patients 7, 10 and 11 (two seizure-free) the location of the surgery differed from our defined affected
281 region, and these patients did not show HFOs in the affected (non-resected) region. Patient 12 did
282 not show HFOs in the resected MEG affected area, but was seizure-free.
283
284 Discussion
285 Principal findings

9
286 We detected HFOs in MEG in eight out of twelve patients by using a beamformer spatial filter
287 and virtual sensors. In five patients HFOs occurred multiple times and were thus reproducible. The
288 virtual sensors were less noisy than the physical sensors, which allowed identification of HFOs in
289 virtual sensors that were not discernable in physical sensors. Marked HFOs could also be recognized
290 in the non-filtered signal and in time-frequency plots. In three patients in whom we could not
291 identify HFOs blindly, we were able to identify HFOs at the moments of spikes. HFOs were detected
292 in the affected region more often than in the mirror region and overlapped with other clinical
293 findings. This non-invasive method enables visual identification of areas showing epileptic HFOs.
294
295 Relation to other studies
296 This is the first study that identifies interictal epileptic HFOs in MEG in the time domain.
297 Marking HFOs in the time domain is a time-consuming procedure, but for an experienced eye the
298 identification of artefacts is easiest and most reliable in the time domain (Bénar et al., 2010; Jacobs
299 et al., 2010; Zijlmans et al., 2012).
300 Earlier MEG studies that looked at high frequency components in the time domain studied
301 evoked potentials. These time locked potentials were averaged to increase the signal to noise ratio
302 (SNR) (Curio et al., 1994). Epileptic discharges are spontaneous and therefore more difficult to
303 average. Also, the number of spikes with a HFO in surface EEG is only around 3-6% (Andrade-Valenca
304 et al., 2011; Melani et al., 2013). Studies that did look at spontaneous high frequency epileptic
305 discharges used time-frequency analysis (Miao et al., 2014; Rampp et al., 2010; Tenney et al., 2014;
306 Xiang et al., 2009). It is yet unclear how these events relate to the time-domain HFOs as previously
307 defined using EEG and intracranial EEG (Jacobs et al., 2012; Worrell et al., 2012; Zelmann et al.,
308 2009). However, the time-frequency plots presented in this study, revealing events that
309 corresponded in time to HFOs identified in the time domain, are comparable to the time-frequency
310 plots shown by others (Miao et al., 2014; Rampp et al., 2010), with HFOs of short duration and a
311 limited frequency bandwidth. In contrast, a recent MEG study by Tenney and colleagues (Tenney et
312 al., 2014) revealed high frequency components during childhood absence seizures, yet these events
313 in their time-frequency plots had a duration of more than one second, which is much longer than the
314 interictal HFOs detected in this and previous studies (Jacobs et al., 2012; Worrell et al., 2012;
315 Zelmann et al., 2009).
316
317 Strengths and weaknesses of the study
318 The application of beamformer virtual sensors enabled us to discern HFOs that could not be
319 identified in the physical sensor data. The beamformer algorithm acts as a spatial filter for activity in
10
320 a target region and therefore attenuates noise as well as the contribution from other (remote) active
321 brain regions. However, the reconstructed virtual sensors have limited spatial resolution that
322 depends on the SNR of the raw data, the depth, position and orientation of the reconstructed
323 sources (Hillebrand and Barnes, 2002), and the geometry of the head. This can result in picking up a
324 spike or HFO at some distance from the real source (Barnes et al., 2004; Wennberg and Cheyne,
325 2014). The inter-sensor distance of 1 cm was chosen based on trial-and-error and provides a tradeoff
326 between the area covered by the virtual sensors and the sensitivity to pick up low-amplitude events.
327 The resolution of the virtual sensors was determined by calculating the correlation of the
328 beamformer weights (Appendix A)(Barnes and Hillebrand, 2003). We found a spatial resolution in the
329 range of 1 cm.
330 The spherical volume conductor model we used, could have the disadvantage of spatial
331 mislocation and/or reduction of the SNR (e.g. Hillebrand and Barnes, 2003, 2011). Because we can
332 see HFOs in the virtual sensors that were not discernable in the physical sensors, we believe that the
333 modelling errors were negligible.
334 The proposed method is a proof-of-principle that we can find MEG HFOs, however it is not
335 suitable for clinical application yet. We chose a region of interest (ROI) based on the location of
336 spikes in the physical MEG sensors to increase the chance of finding HFOs. Comparing our results to
337 the surgical area, it seems this ROI was imperfect in two patients (patient 10 and 11). If any HFOs or
338 spikes would have occurred outside our ROI, we would have missed them. More precise validation of
339 the epileptogenic area found with MEG HFOs is needed in the future. When the workload of visual
340 analysis is reduced by an automatic detection algorithm, it will be possible to place more virtual
341 sensors throughout the brain with high spatial sampling, for example in each voxel of the MRI, or
342 based on an atlas (Hillebrand et al., 2012). This way we can take advantage of the good spatial
343 resolution of beamformers in comparison to some alternative imaging approaches (Lu et al., 2014).
344 Automatic placement of virtual electrodes and automatic HFO detection would make our HFO
345 analysis in MEG easy to use in a clinical setting.
346 We chose to use the high frequency component of the epileptic spikes to calculate the data
347 covariance, because we assumed that these were the moments that we were most likely to find
348 HFOs. Filtering of sharp transients may lead to oscillatory artefacts (Bénar et al., 2010). Spikes that
349 yield 'false' HFOs have to be unnaturally sharp and would therefore not be marked as spikes but
350 considered as artefacts. We only marked spikes of which we were convinced they were true spikes,
351 so the amount of false HFOs due to our method is likely to be small. The choice of using the high pass
352 filtered spikes as data covariance may have introduced bias towards localizing spike-related
353 oscillations, but we did not find support for this when we reviewed the unfiltered data, as HFOs were
11
354 visible. The time-frequency plots of the marked HFOs showed a narrow band ‘blob’ in the high
355 frequencies, distinct from the spike, which pleads against these HFOs being filter artefacts of spikes.
356 Moreover, false ripples are expected to be centered around the sharp peak of the spike. This was not
357 the case for the HFOs in Figures 1 and 4: these HFOs seem to precede the sharpest component of the
358 spike. This is in accordance with our findings for non-invasive EEG HFOs, where 64% of the HFOs
359 precede the spike (van Klink et al., 2015).
360 The spatial extent of the HFOs in the affected area was not much smaller than the spatial
361 extent of the spikes in some patients, in contrast to observations in invasive recordings. Still, HFOs
362 were found almost exclusively in the affected region, while spikes were found bilaterally. When
363 taking a closer look, the distribution of HFOs shows a certain ‘hot spot’ of a few virtual sensors with
364 more HFOs than others, while this is less clear for spikes. The larger-than-expected spatial extent of
365 HFOs might be due to the spatial resolution of the beamformer analysis, or due to under sampling.
366 Future studies with higher spatial sampling could reveal whether the spatial extent of the spikes and
367 HFOs indeed differ, and whether detection of these differences, if they exist, are so small that they
368 require improvements in spatial resolution of this non-invasive approach (Hillebrand and Barnes,
369 2011).
370 We excluded patients without epileptic spikes in physical sensors in our method, but
371 theoretically they might show HFOs. The decreasing sensitivity of MEG with depth might be a reason
372 why some patients do not show MEG spikes on physical sensors (Hillebrand and Barnes, 2002).
373 Spikes from deep sources can be made visible using virtual sensors (personal communication,
374 Appendix Figure B.1.), which suggests that picking up HFOs might also be possible in these cases.
375
376 (Clinical) relevance of the study
377 A good overlap was found between the location of HFOs and most prominent spikes, and a
378 similar overlap with SOZ, MRI abnormalities, PET, SPECT. Successful surgery could confirm that these
379 HFOs probably represented the area of epileptogenic tissue. We focused on patients with multiple
380 HFO times and should be careful interpreting non-reproduced single events in this kind of artifact-
381 prone signal. Almost all patients showed some epileptic spikes in the mirror region, while the clinical
382 analysis showed a single focus in all patients except patient 6. The conclusion of the presurgical
383 investigations was that patient 6 might suffer from idiopathic generalized epilepsy, which could
384 explain the bilateral epileptic spikes and HFOs. In the other patients the epileptic spikes in the mirror
385 region could have spread from the affected region. The sensor with the highest amplitude spike is
386 considered important for clinical analysis, while we did not consider spike amplitude and marked all
387 spikes for our study. HFOs were found almost exclusively in the affected region, thus covering a
12
388 smaller area than spikes. This might indicate that HFOs are more specific for the epileptogenic zone
389 than spikes. This would be in accordance with EEG and intracranial EEG findings (Jacobs et al., 2009;
390 Melani et al., 2013). The large number of sensors in MEG compared to clinical EEG results in a better
391 spatial resolution, and enables the use of beamforming to place virtual sensors in a region of interest.
392 As described above, an automatic detection algorithm for HFOs in MEG would allow us to utilize the
393 good spatial resolution of MEG even further.
394 The SNR seems to play an important role in the ability to identify HFOs in MEG, maybe even
395 more so than for EEG (von Ellenrieder et al., 2014; Muthukumaraswamy, 2013). By specifically
396 looking at the moments that spikes occurred, 290 additional HFOs at 68 HFO-times could be marked
397 in seven of the twelve patients. This doubled the number of HFOs and tripled the number of HFO-
398 times compared to only blind marking. We improved the SNR of the raw MEG data by application of
399 the tSSS filter and the beamformer (Hillebrand et al., 2013). Optimization of noise reduction
400 techniques for high frequencies could further improve the SNR and increase the yield of HFOs (Helle
401 et al., 2012).
402 With the method proposed in this article we show that it is possible to find HFOs in MEG in
403 the time domain, which overlap with the clinically expected epileptic area. When we can automate
404 the detection we can apply this method to a larger population and compare with intracranial and
405 surgical results to validate our findings. Simultaneous recording with EEG/MEG and intracranial EEG
406 is needed to establish whether HFOs in those techniques are the same events (Dubarry et al., 2014).
407 HFO analysis could also benefit patients with epilepsy who are not a surgical candidate, for example
408 to monitor the effect of medication (Kobayashi et al., 2015; Zijlmans et al., 2009). Ideally, the HFO
409 area should be found without the need for guidance by epileptic spikes. Increasing the SNR of the
410 raw MEG data would further aid HFO analysis. Addressing these issues through optimization of the
411 MEG data processing and an automatic detection algorithm for HFOs could make HFO analysis, and
412 thereby non-invasive delineation of the epileptogenic zone, available for a large group of patients
413 with epilepsy.

414
415

13
416 Conflict of Interest
417 None of the authors have potential conflicts of interest to be disclosed.
418
419 Acknowledgements
420 The authors would like to thank Ida Nissen for help with patient selection, and the MEG technicians
421 at the VUmc, Nico Akemann, Marlous van den Hoek, Karin Plugge, Peterjan Ris, Irene Ris-Hilgersom
422 and Ndedi Sijsma, for the recording of the MEG data and/or the clinical MEG analyses. N.E.C. van
423 Klink is supported by the Dutch Brain Foundation fund (number 2013-139) and the Dutch Epilepsy
424 Foundation fund 15-09. M. Zijlmans is supported by the Rudolf Magnus Institute Talent fellowship
425 2012 and ZonMW veni 91615149.
426
427
428

14
429 Appendix A. Correlation of beamformer weigths.
430
431 The spatial resolution of beamformer reconstructions is determined by the correlation between the
432 beamformer weights for the virtual sensors (Barnes and Hillebrand, 2003). We calculated
433 correlations between the weights for all virtual sensors, and also specifically compared the weight
434 correlations between virtual sensors with and without HFOs.
435
436 Correlation between weights for all virtual sensors
437 Figure A.1 shows the correlation coefficient for all virtual sensors with all other virtual sensors.
438 Correlations with sensors within the same hemisphere were higher than with sensors in the
439 contralateral hemisphere. It appeared that neighbouring virtual sensors, one step (i.e. 1 cm) apart in
440 the star-shaped configuration, showed a higher correlation with each other than with sensors at
441 further distance. (Correlation with neighbours: r=0.83, with others: r = 0.28. t(11) = 62.5, p<0.001).
442
443 Correlation between weights of virtual sensors with and without HFOs
444 A high correlation between virtual sensors could explain the existence of an HFO on several virtual
445 sensors. We therefore calculated the correlations between virtual sensors with HFO and non-HFO
446 neighbours, which did not show a significant difference (rneighbour with HFO=0.70, rneighbour without HFO=0.69:
447 t(7)=0.22, p=0.83). Virtual sensors at distance with or without HFO did not show a significant
448 difference in correlation either (rdistance with HFO=0.32, rdistance without HFO=0.25: t(7)=0.78, p=0.46).
449
450 Spatial resolution
451 The distance between virtual sensors was chosen by trial and error at a distance of 1 cm. The
452 correlation of the beamformer weights showed a high correlation with neighbouring sensors at 1 cm
453 distance, and a lower correlation with sensors at larger distance. This implies that the spatial
454 resolution of our beamformer was between 1 and 2 cm. For this specific purpose to find HFOs, the
455 value of correlations of weights can be questioned, because the HFO is only a very small part of the
456 signal. The fact that we identified HFOs on only 2 or 3 virtual sensors at the same time reinforces the
457 assumption that the spatial resolution was in the 1 cm range.
458
459
460 Figure A.1. Mean of all absolute correlations between virtual sensors. Virtual sensors 1 up to 35 were located in
461 the left hemisphere, 36 up to 70 were in the right hemisphere. Correlations between virtual sensors in the
462 same hemisphere were higher than correlations with sensors in the contralateral hemisphere.

15
463 Appendix B.
464
465 Figure B.1. Example of spikes in virtual sensors (patient 5). Virtual sensors (upper two traces) show spikes (grey
466 area) that were not visible in the physical sensor data (bottom traces).
467
468

16
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569

570

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571 Figure Legends
572 Figure 1. Example of physical sensors (A) over the same region as where virtual sensors were placed (B)
573 (patient 9). Epileptic spikes are easier to identify in the virtual sensors (B.1) than in the physical sensors (A.1).
574 The data segment around the spike (grey section in the left panels) is displayed in the middle and right panels.
575 In the middle panel the segment is stretched in time, and the 80Hz high pass filtered signal is displayed on the
576 background. The right panel shows the 80Hz high pass filtered signal again. In the 80Hz filtered virtual sensors
577 (B.1, right) the HFOs can be clearly identified (underlined) and they can also be observed in the unfiltered data
578 (B.1, middle). Note that these HFOs were not discernable in the 80Hz filtered physical sensors (A.1, right). (C)
579 The time-frequency plot of the virtual sensors shows the HFO in sensors VIRT49 up to VIRT53. Neither spikes
580 nor HFOs were visible in the contralateral physical (A.2) and virtual sensors (B.2, D).
581
582 Figure 2. Thirty-five virtual sensors (VS) were positioned in each hemisphere to fill a star-shaped configuration
583 of 6cm diameter. Red dots indicate virtual sensors; grey dots are sensors in a deeper or more superficial layer
584 of the VS group. The localized MEG spikes are shown in green. For the affected region, the backbone of the VS
585 group consists of a 3D cross centered on the location of a localized spike and with arms of 3 cm length. At one
586 centimeter from the center, four sensors were placed and at two centimeter from the center another four
587 sensors are placed to form the diagonals of the star. As a control, another 35 sensors were placed at the
588 homologous position in the contralateral hemisphere.
589
590 Figure 3. Example of high frequency activity co-occurring with a spike in the affected region that was not
591 marked as HFO. A) The spike in the grey section in the unfiltered data (left) was extended in time (middle) with
592 the 80Hz high pass filtered signal in the background. The 80Hz high pass filtered is shown in the right panel
593 again. The filtered data show irregular morphology. This was considered noise. B) The time—frequency plot of
594 the channels in A show inconsistent high frequency activity. C) Again the spike in the grey section in the
595 unfiltered data (left) was extended in time (middle) with the 80Hz high pass filtered signal in the background,
596 and the 80Hz high pass filtered in the right panel. The spike was considered too sharp to be a true spike, and
597 therefore considered an artefact. The filtered data show a sharp and short oscillation, suspect for artefact. D)
598 The time-frequency plot of the channels in C show broad band high frequency activity, which arises because of
599 filtering of the sharp transient.
600

601 Figure 4. Example of additionally marked HFOs on a spike (patient 3). After a first round of blindly marking
602 HFOs, without knowledge of the spikes, additional HFOs were marked at the latency of a spike. These HFOs did
603 not differ enough from the noisy background to be reliably marked ’blindly’, but were likely to be true HFOs
604 because of the co-occurrence with a spike. (A) The spike in the left panel (grey area) is expanded in time in the
605 middle panel, with the 80Hz high pass filtered signal in the background, and the 80Hz high pass filtered again in
606 the right panel. HFOs are underlined. (B) The mirror region shows a sharp wave at the time of the spike in A.

20
607 There is no HFO in the mirror region. (C) The time-frequency plot of the virtual sensors shows the HFO in
608 sensors VIRT60, and VIRT62 up to VIRT64.
609
610 Figure 5. Example of HFOs marked during blind marking (patient 8), which did not co-occur with a spike in the
611 affected region (A) or the mirror region (B). The grey area in the left panels in A and B is expanded in time in
612 the middle panel, with the 80Hz high pass filtered in the background, and 80Hz high pass filtered in the right
613 panel. (C) The time-frequency plot of the virtual sensors shows the subtle HFO in sensors VIRT36, VIRT37, and
614 VIRT40 up to VIRT42. For other latencies, these sensors also showed HFOs that did co-occur with spikes.
615

21
Tables
Table 1. Patient characteristics, showing the MEG affected region (side/location), HFO location, MRI findings, interical and ictal EEG findings, PET and ictal SPECT findings,
pathology and surgery information, and the duration of the registration. HFO location between brackets means only one HFO-time on this side (table 2). M: male, F: female,
L: left, R: right, SEGA: subependymal giant cell astrocytoma, FCD: focal cortical dysplasia, MTS: mesiotemporal sclerosis, NA: not available
MEG affected Interictal EEG Ictal EEG Duratio
Gender/age HFO location MRI PET SPECT Pathology Surgery
# side/location abnormalities onset n (min)
1 M/7 L/Temporal - No abnormalities Possible frontal focus, No No NA No surgery No surgery 14,6
probably R lataralisation abnormaliti
or localisation es
2 M/15 R/Temporal R Multiple cortical tubers, R fronto tempero R, not NA R Tuber, no R neocortico- 15
decreased grey and basal localizing temporal MTS amygdalahippocampecto
white matter my, Engel 1A, 20 months
differentiation R post OR
temporal
3 M/16 R/Temporal R No abnormalities R anterior temporal R temporal R temporal R Normal R temporolobectomy with 14,5
temporal tissue amygdalahippocampecto
my, Engel 1D: one seizure
after stop AED, 11 months
post OR
4 F/16 L/Temporoparietal (L) Minimal white matter L abnormalities R or L in No L No surgery No surgery 15.,0
malformation R frontal different abnormaliti temporal
seizures. es
5 F/17 R/Frontocentral (L and R) No abnormalities Frontocentral midline, Frontocentral R central NA No surgery No surgery 14,7
probably more L
6 M/10 L/Frontal L and R Arachnoidal cyst L Bilateral frontal and NA NA NA No surgery No surgery: suspect for 15,2
temporal generalized late onset idiopathic
generalized epilepsy.
Seizure free with VPA
7 M/6 L/Temporal - Multiple cortical tubers L temporal, more L posterior NA NA SEGA + Resection of growing SEGA 5,7
+ SEGA R near posterior temporal tuber 3rd ventricle + tuber R
intraventricular foramen frontal, Engel 4B, 3
months post PR
8 M/6 R/Parietal R Multifocal gliosis, R R central paramedian R central R frontal or NA No surgery No surgery 13,6
more than L paramedian parieto-
occipital

22
9 M/12 R/Temporal R (and L) MTS R suspect R hemisphere, most R centro- R temporo- R No surgery No surgery 16,5
posterior temporal temporal parieto- temporo-
occipital parietal
10 M/14 R/Temporal (L) FCD R posterior temporal R posterior R posterior NA FCD 2A R occipitotemporobasal 15,2
temporal temporal resection, Engel 1A, 12
months post OR
11 M/12 R/Frontocentral - Ventricular cyst R frontal R frontal and R temporal R anterior NA MTS Wyler R anterior 15
+ MTS R centroparietal diffuse temporal 2 temporolobectomy with
amygdalahippocampecto
my, Engel 1A, 4 months
post OR
12 F/29 L/Temporoparietal - Arteriovenous fistula L No interictal L frontal and NA NA Vessel Leasionectomy of fistula, 15
parietooccipital epileptiform midline sclerosis unable to resect seizure
discharges focus due to speech
arrest, Engel 1A, 13
months post OR

23
Table 2. The number of times when a spike (STime) or an HFO (HFOTime) was marked, and the total number of
marked spikes (S), blind marked HFOs (HFO), and additional HFOs marked by specifically looking at the spike-
times (Add HFO). Between brackets is the number of virtual sensors over which the events spread. The
latencies at which an HFO occurred in virtual sensors were reviewed in the physical sensors to see whether the
HFO was also discernable in the physical sensors (Also in Ph) or not. Virtual sensors showed an HFO that could
not be identified in the physical sensors at 61 out of 78 time points.

Affected Mirror
STime Add HFOTime Also STime Add HFOTime Also
# (#Chann) S HFO HFO (#Chann) in Ph (#Chann) S HFO HFO (#Chann) in Ph
1 14 (21) 79 0 0 0 6 (18) 32 0 0 0
2 82 (35) 871 17 24 7 (18) 0 5 (29) 45 0 0 0
3 122 (35) 1463 0 15 4 (14) 0 23 (27) 176 0 0 0
4 31 (35) 253 0 2 1 (2) 0 26 (35) 199 0 0 0
5 51 (34) 434 0 4 1 (4) 0 17 (34) 169 0 2 1 (2) 0
119
6 71 (35) 1613 86 113 15 (32) 9 59 (35) 0 50 7 (26) 2
6
7 11 (29) 189 0 0 0 0 0 0 0 0
1042
8 10884 44 33 19 (19) 2 46 (28) 229 0 0 0
(35)
9 74 (35) 1416 136 39 21 (34) 4 22 (33) 149 0 8 1 (8) 0
10 8 (23) 41 0 0 0 6 (29) 53 2 0 1 (2) 0
11 27 (29) 165 0 0 0 5 (33) 76 0 0 0
12 48 (34) 584 0 0 0 28 (30) 379 0 0 0

24
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure A1
Figure B1