Fig. 2. In vitro electrophysiological characterisation of scanless two-photon voltage imaging in cultured CHO cells.

a (i) Schematic representation of the protocol used to prepare JEDI-2P-Kv expressing CHO cells (“Methods” section). (ii) Scanless 2P voltage imaging was performed using temporally focused (TF) GPC (Generalized Phase Contrast), Gaussian or holographic (CGH) spots. (iii) Transmitted light image of a patched CHO cell (left) and representative confocal image of a different JEDI-2P-Kv expressing CHO cell (right) (n = 41 cells, 19 independent transfections). Scale bars represent 10 µm. b Data from protocol 1 (“Methods” section, Supplementary Table 5) used to assess the performance of each illumination modality (100 Hz acquisition rate, power density 0.88 mW µm⁻²). The electrophysiological (ephys) command voltage is plotted in black with relevant holding potentials indicated. The red bar represents the illumination epoch. (ii–iv) Average fluorescence responses acquired using each modality. Results from single trials from independent cells are also plotted in grey. Responses are reported as (-%∆F/F₀ (n = 9 (Gauss), 15 (CGH), 17 (GPC) cells; from 2 (Gauss, CGH) or 3 (GPC) independent transfections). c Representative imaging data acquired during scanless 2P voltage imaging experiments on JEDI-2P-Kv expressing CHO cells at 100 Hz, (i) and 1 kHz, (ii) (power densities 0.88 and 1.11 mW µm⁻² respectively). Left: single frames, middle: temporal average of all frames, right: corresponding pixel weights (all normalised) used to generate the final fluorescence traces (“Methods”). (d-e) Violin plots (shaded) summarising the performance: -%∆F/F₀ (d) and SNR (e), of each scanless 2P voltage imaging modality at different illumination power densities (0.66–1.55 mW µm⁻², n = 8 (Gauss), 13 (CGH), 12 (GPC) cells, 2 independent transfections per modality). Results presented in d and e were acquired using protocol 2 (“Methods” section, Supplementary Table 5). In every case, a coloured point represents a single measurement from an individual cell, a black cross is located at the population mean and the coloured bars (adjacent) depict the interquartile range. f (i) Electrophysiological command voltage for protocol 3 (“Methods” section and Supplementary Table 5) plotted in black with relevant holding potentials indicated. The red bar represents the illumination epoch. (ii–iv) Corresponding scanless 2P imaging data acquired using each parallel illumination modality. Results from single trials from independent cells are also plotted in grey (power density: 1.33 mW µm⁻², n = 8 (Gauss), 11 (CGH, GPC) cells, 2 independent transfections per modality). Source data are provided as a Source Data file.