Fig. 4: Activation of ChR2 under different stimulation conditions.
From: Active pixel power control for crosstalk-free all-optical neural interrogation
a The activation probability of a ChR2 molecule under stimulation by two-photon raster scanning at different powers of a defocused laser (Simulated N.A. = 0.9). The dashed lines correspond to the stimulation times for 100 μm, 200 μm, and 400 μm FOVs using a resonant scanner with an 8 kHz resonance frequency. The results account for the attenuation caused by the sample tissue (see “Methods”). For simulation results that do not consider attenuation for in vitro experiments as reference, see Fig. S14. b Illustration of raster scanning through the entire focal plane at a distance z from the cell equator plane (orange). Green circle with red outline: soma. c Simulated photocurrents stimulated by raster scanning at various distances Z related to the equator. d The spikes value (mean ± s.d.) of target neuron with an extra scanning plane outside of cell equator (n = 24 neurons, 15 fish, Friedman test with Dunn’s multiple comparisons test, two-sided; 0 µm: 7.68 ± 4.57, 2 µm: 6.75 ± 2.97, 5 µm: 6.54 ± 3.16, 10 µm: 5.33 ± 2.44, 14 µm: 4.79 ± 2.48, OFF: 4.35 ± 1.85). Black dot and black line: simulated results in (c) and fitted to spike value. e Illustration of the volumetric calcium imaging (10 planes) experiment with plane gap of 2–14 µm. The equatorial plane of target neuron located at the 6th plane. f The spikes value (mean ± s.d.) of target neuron with an extra scanning plane outside of cell equator (n = 30 neurons, 10 fish, Friedman test with Dunn’s multiple comparisons test, two-sided; 2 µm: 6.79 ± 4.46, 5 µm: 5.81 ± 4.51, 8 µm: 5.08 ± 3.18, 10 µm: 4.76 ± 2.95, 14 µm: 4.61 ± 2.57, OFF: 4.21 ± 2.42). g Simulated photocurrents evoked by raster scanning at various distances Z from the equator under different imaging conditions. h The spikes value (mean±s.d.) of target neuron at 5 mw whole cell imaging, 15 mw sub-soma imaging, and 15 mw whole cell imaging. (n = 12 neurons, 5 fish, one-way ANOVA with Tukey’s multiple comparisons test, two-sided; 5 mW whole cell: 4.146 ± 2.09, 15 mW sub-soma: 3.91 ± 1.67, 15 mW sub-soma: 8.47 ± 4.46).
