Fig. 3. Photoactivation under cyclic-FLiT.

a Photostimulation of a group of neurons under steady and cyclic-illumination. A soma-targeted light pattern encoded by a single hologram can be used to photoactivate a group of neurons either under steady illumination of power $P_{std}$ and duration $t_{dw}$ (black line, top) or under cyclic-illumination of $N_{cyc}$ pulses at power $P_{cyc}$, period $T_{cyc}$ and pulse duration $t_{cyc}$ (red line, middle). Simulated photocurrents generated in a ST-ChroME-expressing neuron are shown under steady (black, bottom) and cyclic (red, bottom) illumination when $P_{cyc}=P_{std}\sqrt{T_{cyc}/t_{cyc}}$ ($P_{std}=0.05$ mW/µm²; $P_{cyc}=0.05\sqrt{20}$ mW/µm²; $T_{cyc}=20t_{cyc}$; $t_{cyc}=50\mathrm{\mu }\mathrm{s}$; 1030 nm). b Conceptual scheme of simultaneous photostimulation of multiple groups of neurons under cyclic-FLiT. The LC-SLM is tiled in multiple holograms φ_i (here from φ₁ to φ₅) each encoding for distinct soma-targeted multicell light patterns encoding for different groups of cells (here from Group 1 to Group 5). The illumination beam is switched across the holograms such that the light is sequentially redirected to $H$ = 1/D holograms, and the same cyclic photoactivation process is enabled sequentially on the different light patterns. The scheme displayed is meant to represent H groups of spots; their number is here limited to 5 for presentation purposes only. c Measurements of the switching time to sequentially illuminate all holograms at constant rate from φ₁ to φ_n by driving the galvanometric mirror (GM) with a staircase voltage input. d GM voltage input (black line) and corresponding position of the incoming beam on the LC-SLM (red line) when GM is driven as depicted in (c). e Pulse-width dwell time of each hologram φ_i of the LC-SLM while GM is driven as depicted in (c) and φ_i only encodes an individual spot in the middle of the PD. Horizontal black and gray lines indicate the mean and SD dwell time over all holograms, respectively. f Mean latency and jitter of the light-evoked APs obtained under cyclic-FLiT (brown) and conventional illumination (green) for different number of holograms (n = 8 cells; data are shown as mean $\pm$SD). For conventional holography: total illumination time $t_{\exp }=t_{dw}$= 5 ms; For cyclic-FLiT: total illumination time $t_{\exp }=$ 5 ms; $t_{dw}=t_{\exp }/H$). Data are nonsignificantly (ns) different between the two illumination protocols (P value = 0.23 and P value = 0.31 for latency and jitter, respectively; Mann–Whitney test, two-tailed). g Ratio of the powers needed to trigger a light-evoked AP with 5 ms total time of illumination under steady P_std and cyclic-illumination P_cyc illumination and different number of holograms H. Different colors indicate different cells (n = 5 cells; data are shown as mean $\pm$SD). Red asterisks represent the theoretical expected $\sqrt{H}$ ratio value. Top inset: Threshold power to activate the cells under steady illumination with $t_{dw}=5\mathrm{ms}$. Bottom inset: representative light-evoked APs under steady (black) and cyclic (red) illumination of duration 5 ms. Source data are provided as a Source Data file.