Supplementary Figure 5: In Vitro Voltage Imaging.

a) Hippocampal culture labeled with RhoVR.pip.sulf. Dashed lines denote fields of view recorded by fast widefield and SLAP imaging. b) ΔF/F₀ traces for the neuron outlined in (a) using two-photon SLAP without a dynamics prior (τ=0, top) or one-photon widefield imaging (bottom), in separate trials. Suprathreshold field stimuli (90 V/cm; black arrowheads) were applied to evoke action potentials. (right) Zoom of the red boxes in b. (c-e) FOV containing multiple neuron somata. Low-amplitude field stimuli (50 V/cm; black arrowheads) were applied to produce asynchronous spike patterns. Widefield imaging frame rate was limited to 400 Hz due to the size of the FOV. e) Scatterplot of SLAP responses of the two cells, demonstrating distinct patterns of firing in the two neurons. Amplitudes were the sum ΔF/F₀ in the 20 ms following each stimulus, divided by the mean of the 3 largest amplitudes per recording. d’ values were calculated by fitting two Gaussians of equal variance (dotted lines) to the marginal activity distribution for each neuron (histograms). The cell outlines in (a,c) denote ROIs used to generate traces, by pixel-averaging (widefield) or frame-independent nonnegative unmixing (SLAP). Imaging power: 31 mW two-photon, 0.7 mW one-photon. These powers were the maximum that did not result in noticeable photobrightening of the voltage indicator, which occurs at higher excitation powers in both modalities. (a-d) We recorded 16 fields of view with similar results.