Fig. 6. In vivo high-resolution volumetric voltage imaging of sparsely labeled neurons in Drosophila at 500 vps.

a–c, 3D rendering volumes and enlarged MIPs of PPL1 dopamine neurons at a depth of 15 μm in Drosophila brain (MB065B-GAL4 > 20×UAS-pAce), obtained by LFM (a), VsLFM (b) and sLFM (c). d, Average temporal traces extracted from two different regions in the raw light-field images. e, Left, temporal traces extracted from the manually selected region in b and c for VsLFM and sLFM, with the black circles marking the identified spikes. Right, corresponding average waveforms for the spikes. The data of sLFM and VsLFM were collected sequentially on the same Drosophila, therefore the spontaneous voltage activities occurred at different time stamps. f,g, Comparisons of the temporal FWHMs (f) and amplitudes (g) of the spikes between VsLFM and sLFM in the same selected region as e. The center line represents the median, the box limits represent the lower and upper quartiles, and the whiskers represent 1.5-fold the interquartile range. P values were calculated using the two-sided paired t-test. P = 1.77 × 10⁻¹⁵ (f) and P = 8.14 × 10⁻¹¹ (g). n = 5 for the sLFM results and n = 26 for the VsLFM results, where n represents the number of identified spikes. h, 3D rendering volume of PPL1 dopamine neurons at a depth of 100 μm in another Drosophila (MB065B-GAL4>20×UAS-pAce) obtained by VsLFM, with the enlarged time-coded MIPs. Different colors represent the peak instants of the voltage signal for every pixel during 2.4 ms. i, Voltage spikes extracted from two regions in the PPL1-α‘2α2 neuron, showing a 2 ms delay in action potentials. j, Upper row, odor-evoked voltage traces extracted from the region marked by the white dashed circle in h. Bottom row, corresponding time-dependent firing rates. The gray rectangles indicate the time window when we applied the 3% benzaldehyde (BEN) stimulus. Scale bars, 30 μm (a–d, h).