Figure 3.

AP threshold with standard femtosecond Ti:Sapphire oscillator and spatial specificity of 2P holographic stimulation of ReaChR. (A) Widefield fluorescence image of L2/3 ReaChR-expressing neuron filled with 20 μM Alexa 594. Scale bar: 15 μm. (B) Voltage response (single recording) to a 10 ms long (red line) 2P holographic stimulation with a standard femtosecond Ti:Sapphire oscillator at 950 nm using a circular spot on the cell soma (1 in panel A, 15 μm diameter) for increasing LPDs. (C) Average voltage response (n = 3 repetitions) to a 10 ms long (red line) 2P holographic stimulation with a standard femtosecond Ti:Sapphire oscillator at 950 nm on numbered circular spots (15 μm diameter) in panel A. LPD: 0.2 mW/μm². (D) Distribution of average voltage response (n = 3 repetitions, 25 spots, 3 cells) to a 10 ms long (red line) 2P holographic stimulation with a standard femtosecond Ti:Sapphire oscillator at 950 nm on cell processes. Distance from the soma from 15 to 100 μm, as shown in panel A. LPD corresponding to the one used to reach the AP threshold for 2P stimulation on the cell soma for each cell (0.15 to 0.25 mW/μm²).