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. 2021 Jun 23;41(25):5372–5385. doi: 10.1523/JNEUROSCI.2765-20.2021

Figure 1.

Figure 1.

Optical and electrophysiological measure of the somatic AP waveform. A, Schematic representation of the experimental setup; neurons expressing a GEVI were held in whole-cell current clamp while simultaneously imaging a segment of the somatic membrane at room temperature. The stimulation protocol consisted in a time-locked current pulse delivered through the patch pipette (I) to evoke a single AP, while subjecting the cells to LED or laser illumination (light) and high-speed camera acquisition at 3.2 kHz. The protocol was repeated 20 times, and the resulting recordings were averaged. B, C, Representative example cells expressing Ace-mNeon (B) and Archon2 (C), with selected ROIs of the somatic membrane for optical trace analysis shown in red. B′, C′, Reference Ephys (left) and single-trial optical (center) recordings of evoked somatic APs acquired from the example cells in B and C, respectively. Overlay of voltage and optical somatic AP recordings (right) for Ace-mNeon (green) and Archon 2 (orange). D–F, Evoked AP width (D), decay time (E), and amplitude (F) were measured simultaneously with Ace-mNeon and reference Ephys recordings in the same cells to assess accuracy of GEVI measurements. G–I, Evoked AP width (G), decay time (H), and amplitude (I) were measured simultaneously with Archon2 and reference Ephys recordings in the same cells to assess accuracy of GEVI measurements. J, SNR of optically recorded APs with the two GEVIs; *p < 0.05; Kruskal–Wallis tests. K, Steady-state photocurrent induced in response to GEVI illumination with the corresponding light source (505 nm LED at 10 mW/mm2 for Ace-mNeon; 635 nm laser at 2.6 W/mm2 for Archon2); ***p < 0.001; one sample Wilcoxon test. L, M, Absolute AP width measurement error (L) and decay time measurement error (M) recorded with Ace-mNeon and Archon2 relative to reference Ephys recordings. Ace-mNeon, N = 16 cells; Archon2, N = 19 cells. R, Pearson's correlation coefficient; L, M, Mann–Whitney tests; ***p < 0.001, **p < 0.01. All measurements performed on averages of 20 repeats.