Fig. 2. Tuning of neuronal activity in targeted neurons by hybrid-FLiT.

a Conceptual scheme of hybrid-FLiT. The LC-SLM is tiled in different regions each encoding different phase masks. In the present example, phase mask φ_A and φ_B encode for group of spots A and B, while phase mask φ_AB encodes for a comprehensive pattern including group A and group B. By steering the beam vertically across the phase masks with predetermined dwell-times and illumination intensities per each mask, it is possible to set arbitrary delays of activation between groups of spots. In the illustrated example, the illumination dwell time is t_φA, t_φAB, t_φB, and the illumination power is P_A, P_A + P_B, P_B on the holograms φ_A, φ_AB, φ_B, respectively. On comprehensive phase mask φ_AB, the distribution of intensity must be computationally set to maintain an amount of power P_A and P_B on subgroup A and B, respectively. Overall, this scheme yields an activation time t_φA + t_φAB for group A, t_φAB + t_φB for group B and a delay of activation between group A and group B δt equivalent to t_φA. t_sw represents the switching time of the GM unit. Yellow vertical bands indicate the switching time t_sw. The scheme displayed is meant to represent n groups of spots; their number is here limited to 2 for presentation purposes only. b Measurement of the switching time between two adjacent tiled holograms (φ_i and φ_i+1) when galvo moves in one discrete step. A photodiode (PD) is placed in an image conjugate plane while driving the galvanometric mirror (GM) with a small-angle single-step voltage input. c Representative intensity response of the PD when GM is switched from hologram φ₁ (encoding for an individual spot in the middle of PD) to hologram φ₀ (deviating the beam out of the PD) (black line) or, vice versa (red line). SLM was subdivided in 20 holograms. d Switch time calculated as the time taken for the signal to rise/fall between 3% and 97% of the maximum intensity, when the spot is encoded in hologram φ_i and GM is switched from hologram φ_i to φ_i+1 (black symbols) or vice versa (red symbols). Horizontal black line and shaded gray band indicate the global mean and SD switching time, respectively. e Schematics of the experiment for testing the timing of neuronal activity control. Two ST-ChroME expressing patched neurons (cell 1 and cell 2) are photostimulated by using hybrid-illumination of three holograms imposing a tightly controlled delays, $\delta t$, ranging from 0.2 to 3 ms, while measuring the corresponding spiking delay time ${\delta \mathrm{t}}_{\mathrm{AP}}^{\exp }$. Different colors correspond to different delays. Bottom inset: detail of the AP peaks. f Left: Temporal accuracy calculated as the difference between imposed $\mathrm{\delta }\mathrm{t}$ and experimental ${\delta \mathrm{t}}_{\mathrm{AP}}^{\exp }$ delays, $∣{\delta \mathrm{t}}_{\mathrm{AP}}^{\exp }-\delta \mathrm{t}∣$. Circle symbols represent different pair of cells activated with $\delta t$ delays (data are shown as mean $\pm$ SD). Horizontal dashed line and bands of different colors represent the mean and SD at a specific delay $\mathrm{\delta }\mathrm{t}$ as indicated on the left, respectively. Error bars are SD on n = 12 pairs of cells. Right: Global mean temporal accuracy of all pairs of cells shown on the left delayed of $\mathrm{\delta }\mathrm{t}$. Different colors correspond to different delays as indicated on the left. The vertical dashed line and the gray band indicate the average and SD temporal accuracy globally calculated for all pairs of cells (96 ± 114 µs). Data are shown as mean $\pm$ SD. Mean photostimulation power is 36.8 ± 20.9 mW. Illumination dwell time 4–5 ms. In total, 1030 nm illumination has been used. g Temporal accuracy of light-driven random spiking patterns calculated as difference between imposed ${\delta \mathrm{t}}_i$ and experimental ${\delta \mathrm{t}}_{\mathrm{APi}}^{\exp }$ delays of the ith AP pair, $∣{\delta \mathrm{t}}_{\mathrm{APi}}^{\exp }-{\delta \mathrm{t}}_i∣$. Circles from light to dark blue indicate temporal accuracy from subsequent pairs of APs (data are shown as mean $\pm$ SD). The horizontal dashed line and the gray band indicate the average and SD temporal accuracy globally calculated for all pairs of cells (n = 6 pair of cells). Mean photostimulation power is 37.7 ± 21.3 mW. Illumination dwell time 2–5 ms. In all the panels, 1030 nm illumination has been used. Inset: Representative light-driven APs from two double-patched ST-ChroME-expressing neurons (bottom) by imposing a random spiking pattern featuring inter-spike-time intervals ${\delta t}_i$ (top). Source data are provided as a Source Data file.