ICA vs. ROI Signal Extraction

Figure 1: Calculation of Signal-to-Noise Ratio (SNR).
(A) Simultaneous optical and electrophysiological recordings of several cells under several different conditions %(pre-synaptic chemical stimulation, pre-synaptic gap-junction stimulation, spontaneous activity, swimming). (B) Raw intracellular voltage trace. (C) The Voltage trace is down-sampled to the optical frequency based on a method that mimics the optical sampling mechanism. (D) The raw optical trace is shown in red and a polynomial is used to fit the optical trace to the ephys trace (dark red). %The polynomial removes bleaching artifact. (E) The fit optical trace is overlayed on the ideal optical trace. (F) The SNR is calculated as the standard deviation of the Ideal trace divided by the standard deviation of the difference between Ideal and Fit (i.e. the residuals).

Figure 2: Sub-pixel motion correction with ECC.

(A) Raw image of ganglion stained with VSD with two ROIs at the edge of the ganglion. (B) Sub-pixel motion artifact is apparent and large compared with VSD signals. (C) Motion artifact can be removed with ECC image registration algorithm. (D) The registration algorithm performs an affine transformation of the pixels. Each panel corresponds to the warp matrix values over time. (E) The resulting motion artifact that is removed. The separation of blue and black highlights the impact of the skew and rotation values of the affine transformation

Figure 3: Comparison of ROI and ICA based signal extraction.

(A) Raw image with red ROI shown during pre-synaptic stimulation. The optical signal is the average of all pixels within the ROI for each frame. (B) ICA pulls out a component that is manually selected coming from the same cell. The ROI is shown on top to compare localization of component and hand-drawn ROI. (C) The Fit trace is overlayed on the Ideal trace for 4 conditions: ROI only, ROI with Motion Correction (ROI+MC), ICA only, and ICA with Motion Correction (ICA+MC). (D) Raw shown during swim behavior. (E) ICA component. This component shows weight in the bi-lateral pair of cells, as these cells are highly correlated. (F) SNR is compared under the 4 conditions. The 5 mV oscillation are much more clear in the ICA+MC case, even though the SNR increase is fairly small.

Figure 4: SNR comparison of different methods.

(A) The SNR for each method is compared against the ROI method. (B) ICA+MC is compared with ROI+MC as the baseline. (C) The difference in SNR for each trace is plotted as black dots, and the average is plotted as the bar graph. Each method significantly increases SNR (ROI+MC: p = 0.0358, ICA: p = 0.0013, ICA+MC: p=0.0024, t-test). (D) Same as C but with ICA+MC compared to ROI+MC (p=0.004).