Figure 3. Two-photon 3D stimulation of two individual neurons with SLMs.

(a) Left: Two-photon fluorescent image of two C1V1_T-expressing neurons located in the same focal plane, which were patched. Imaging parameters: 940 nm, 15 mW on sample, 20× 0.5 NA objective. Image look-up table is inverted for clarity. An SLM phase mask was calculated to generate one photostimulation laser spot for each cell, and both laser spots were then raster-scanned simultaneously across the cell bodies (Boxes). Photostimulation parameters: 1064 nm, 30 mW per target, ROI 32×32, 2 msec/line. Middle: Whole-cell current clamp recordings from both cells during two-photon SLM photostimulation (black marks). Right: Light intensity generated by different number of SLM targets in similar experiments (see Supplementary Fig. 9 for details; 3–15 measurements per target; error bars are SD). (b) Depth selectivity of SLM photostimulation. Left: Two-photon fluorescent image of two C1V1_T-expressing neurons, located 20 µm apart in depth, which were patched. A single-beam SLM stimulation spot was scanned (box). Imaging parameters as in (a). Right: whole-cell current clamp recordings from both neurons during photostimulation of one of them (black marks; black box in left) with the SLM spot. Photostimulation parameters: 1064 nm, 30 mW in one target, ROI 32×32, 2 msec/line. (c) Same experiment as in (b) but now using a two-dimensional two-beam SLM pattern. Left: Two-photon fluorescent image of two neurons. Imaging as in (a). A new SLM pattern is scanned in the superficial focal plane in the position corresponding to the two cells (boxes). Right: simultaneous dual whole cell recordings during photostimulation (black marks). Photostimulation parameters: 1064 nm, 30 mW per target, ROI 32×32, 2 msec/line. This generates (d) Same experiment as in (c) but with a three-dimensional SLM pattern, which directed two laser beam spots onto both cells simultaneously. Left: Two-photon fluorescent image of both neurons illustrating the simultaneous, multifocal SLM stimulation (boxes). Same imaging parameters as in (c). Right: simultaneous dual whole-cell recordings during photostimulation (black marks). Same photostimulation SLM parameters as in (c). .