Figure 3.

Voltage imaging of AP waveform in axons. A, Schematic representation of the experimental setup; neurons expressing Ace-mNeon or Archon2 were stimulated locally with a bipolar electrode while imaging a fragment of the axonal membrane. Time-locked 1 ms stimulation pulses of 10V were delivered to elicit single APs while subjecting the cells to LED or laser illumination and high-speed camera acquisition at 3.2 kHz. Experiments performed at near physiological temperature (32°C). B, C, Top, Example axons from cells expressing Ace-mNeon (A) and Archon2 (B) with selected axonal ROIs shown in red. Bottom, GEVI recordings obtained from the respective ROIs, showing single trials as well as the averages over sequential groups of 20/50 repeats within the same recording. The arrow indicates the timing of stimulation pulses. D, Average bleaching curve of recordings with Ace-mNeon and Archon2 under exposure with 10 mW/mm² 505 LED light and 2.6 W/mm² laser light, respectively, normalized to initial fluorescence intensity. E, GEVI signal magnitude in response to an evoked AP in the axon, expressed as the absolute value of ΔF/F for the two GEVIs. F, Width of axonal APs recorded with the two GEVIs. G, SNR of axonal APs recorded with the two GEVIs. H, K, Top, Imaging window with selected ROI of the axonal membrane shown in red. Bottom, average Ace-mNeon (H) and Archon2 (K) traces before and after addition of 30 μm 4-ap. I, J, Axonal AP width (I) and amplitude (J) measured with Ace-mNeon before and after addition of 30 μm 4-ap. L, M, Axonal AP width (L) and amplitude (M) measured with Archon2 before and after addition of 30 μm 4-ap. D–G, Ace-mNeon, N = 9 cells; Archon2, N = 6 cells; Mann–Whitney tests; I, J, L, M, Ace-mNeon, N = 6 cells; Archon2, N = 6 cells; Wilcoxon tests; *p < 0.05, **p < 0.01.