Fig 1.
Schematic of the assembled measuring system.
In the middle of the in vitro two-photon measuring chamber the brain slice is placed on a holder mesh. The chamber provides the aCSF circulation near the neural tissue to keep it bioelectrically active. Under the fluid immersed two-photon objective the applied MEA was inserted into the tissue.
Fig 2.
Representative sample of the imaging laser impact on the electrophysiological recordings.
Between the first and the last parts of the measurement, which were recorded without two-photon imaging, photoelectric artefacts of the two-photon imaging laser are observable (A). The recorded data at the moment when the imaging laser was switched on (B, C).
Fig 3.
Filtering and analyzing steps.
The performed filtering and analyzing steps in order to identify the spike clusters and check the spike consistency between the two-photon imaging laser noise free and the laser noisy data (A), the parameter setting algorithm of the applied custom-set comb filter (B) and the result of the parameter setting of a representative filter module (C).
Fig 4.
The absolute value of the frequency spectrum of the electrophysiological recordings.
The fast Fourier transform analysis of the imaging laser generated noise in the electrophysiological recorded data (A). Harmonics below 1200 Hz (C) and at higher frequencies (B) of the laser generated periodical artefacts appeared with high magnitudes. The overlap of the harmonics is observable (B). A part of the rejected frequencies by the custom-set comb filter is shown in yellow (D).
Fig 5.
The applied principal component selection.
Each potential spike was defined with their three principal component before spike sorting: the location of the minimum amplitude value of the spike, and the fifth datapoints before and after the peaks.
Fig 6.
The imaging reveals activities of neuron somas (subfigures 1, 3, 4, 5) and dendrites (subfigure 2) in the vicinity of the microelectrode array.
Fig 7.
Representative sample of the results of the applied filtering algorithm.
The subfigures show the same data as Fig 2 does, prior to filtering (green) and after applying the filtering algorithm (red).
Fig 8.
Representative results of the simultaneous measurement of electrophysiological recordings and two-photon imaging.
The examined electrodes of the applied MEA were in the two-photon imaging window (A). Two examples of sorted spikes recorded by the electrodes shown in part A (B-D). Potential spikes were sorted using three features obtained from the comb-filtered signals (B). The obtained spike waveforms, their averages (C, top) and the autocorrelograms of the thus sorted spikes (C, bottom). The verification of spike stability during laser noise free and the simultaneous two-photon measurements (containing laser noise) is presented in part D. Changes in the shapes of the detected spikes were observed by comparing the averages and standard errors of spike features during the measurements (D, top). The number of the sorted spikes per minute (D, bottom).