Figure 1.

Characterization of currents evoked by 2P holographic stimulation and two-photon excitation spectrum. (A) Simplified scheme of the 2P holographic stimulation microscope. The output beam from an Ytterbium-doped photonic crystal fiber amplifier Laser System was attenuated by rotating a half-waveplate combined to a polarizer cube. The beam was relayed and expanded to cover the surface of an LCoS-SLM (Hamamatsu X10468-07). Zero-order excitation was suppressed by aberrating it with a cylindrical lens. Then the SLM was imaged through a telescope (L1, L2 lenses) at the back focal plane of an IR antireflection coated water-immersion objective (Nikon CFI APO 40X WI NIR NA 0.80) mounted on an upright microscope (Scientifica). (B) CHO cells were voltage clamped at −40 mV and the current response to 2P holographic stimulation (1000 ms, red bar, λ = 1030 nm) with patterns matching the cell shape was recorded. Evoked response for increasing Laser Power Densities (LPDs) in a single cell. Average of n = 3 repetitions. (C) The maximum current (I_max) reached by 2P holographic stimulation at 1030 nm of 4 CHO cells was normalized to the saturation current (I_sat) and plotted vs. LPD. Average of n = 3 repetitions for each cell and LPD. Data recorded above a LPD of 0.2 mW/μm² was not shown as all cells had reached the saturation current. (D) Rise time (T_R, gray dots) and decay time (T_D, black diamonds) for 2P holographic stimulation of a single CHO cell. Average of n = 3 repetitions. T_D and T_R were fit, respectively, by a linear function (black line) and following Equation 4 (in Methods see Section Currents evoked by photo-stimulation of opsins) (gray line). (E) CHO cells were voltage clamped at −40 mV and the current response to 2P Generalized Phase Contrast (GPC) stimulation (400 ms, red bar) was recorded at a wavelength range from 720 to 1030 nm, while keeping the photon flux constant (2.7 × 10²⁶ photons/s/m²). Average of n = 3 repetitions for each wavelength. (F) The maximum current reached by 2P GPC stimulation was normalized to the current generated with stimulation at 1000 nm and plotted vs. excitation wavelengths. A constant photon flux of 2.7 × 10²⁶ photons/s/m² was used. The current generated between 975 and 1030 nm (^*) was significantly larger than currents generated at other wavelengths (p = 0.05, Wilcoxon paired T-test; n = 6 cells). (G) Decay time (T_D, black diamonds) and rise time (T_R, gray dots) for a constant photon flux of 2.7 × 10²⁶ photons/s/m² for 2P holographic stimulation of CHO cells. Average of n = 6 cells. T_R between 975 nm and 1000 nm (^*) was significantly larger than currents generated at other wavelengths (p = 0.05, Wilcoxon paired T-test; n = 6 cells). (H) The 2P cross-section, normalized to its value for excitation at 1000 nm, was plotted vs. excitation wavelengths. The 2P cross-section at 975 and 1000 nm (^**) was very significantly larger than the cross-section at other wavelengths (p = 0.01, Wilcoxon paired T-test; n = 11 cells).