Fig. 1. Detection system with opposite-facing objectives. BE, beam expander; ZB, zero-order blocker; L, lens; M, mirror; obj, objective; S, sample; F, filter; TL, tube lens.

Fig. 2. Characterization of generated holographic patterns for a single target stimulation. (a)–(f) Two-photon intensity distributions in xy (upper) and xz (lower) section of beaded ring of 8, 16, 32, and 64 foci, annular, and disk patterns with 5 μm radius, respectively. Scale bar: 5 μm. (g) Lateral intensity distribution of each focus in (a) and the average lateral intensity of all eight foci, labeled with gray and red lines, respectively. (h) Axial intensity distribution of different patterns shown in (a)–(f). N8, beaded-ring pattern of eight foci (N16, N32, and N64 have similar definitions); A, annular pattern; D, disk pattern. (i)–(k) Two-photon intensity distributions in xy (upper) and xz (lower) section of beaded-ring of 16 foci, annular, and disk patterns with 10 μm radius, respectively. Scale bar: 10 μm. (l) Axial resolution of the beaded-ring, annular, and disk patterns with different radii. D, disk pattern; A, annular pattern, BR, beaded-ring pattern. Five patterns of different radii for each type are generated, and the center of each pattern is 20 μm away from the origin. The data shown in (l) are the mean and standard deviation of axial resolutions of five patterns.

Fig. 3. Characterization of generated holographic patterns for multiple target stimulation in a plane. (a)–(f) Two-photon intensity distributions in xy (upper) and xz (lower) section of beaded-ring, annular, and disk patterns, respectively. (a)–(c) Targets are in the same lateral section (xy), whose centers are from −50 to 50 μm with a lateral interval of 20 μm. (d)–(f) Targets are in the same axial section (xz), whose centers are from −100 to 100 μm with an axial interval of 50 μm. (g), (h) Axial resolution of every pattern in (a)–(c) and (d)–(f), respectively. Scale bar: 5 μm. All pattern distributions in (a)–(f) are obtained under the same excitation power level. D, disk pattern; A, annular pattern; BR, beaded-ring pattern. Five groups of patterns in different distributions (xy or xz) of each type are generated, and the distances from patterns of different groups to the center of the field of view are equal for the same distribution. The data shown in (g), (h) are the mean and standard deviation of axial resolutions of five group patterns.

Fig. 4. Characterization of generated holographic patterns for multiple target stimulation in 3D stack. (a)–(c) Intensity distributions (upper) and maximum intensity projections in xy section (lower) of 10 targets with beaded-ring, annular, and disk patterns, respectively. Radius of each pattern: 5 μm. Stack size: 120  μm×120  μm×240  μm. (d) Statistical analysis of axial resolutions of each pattern in (a)–(c). (e) Statistical analysis of maximal intensities of each pattern in (a)–(c). The central mark of each box in (d), (e) indicates the median, while the bottom and top edges of the boxes indicate the 25th and 75th percentiles, respectively. BR, beaded-ring pattern; A, annular pattern; D, disk pattern.

Fig. 5. All-optical physiology test with different stimulation patterns on a single neuron in vivo. (a) Schematic diagram of the all-optical physiology system. Green: beam for two-photon calcium imaging. Red: beam for two-photon optogenetics. Diagram at lower right: excitation pattern distribution tested by the all-optical physiology system with 1 μm fluorescent beads. D, disk pattern; BR, beaded-ring pattern. Scale bar: 5 μm. (b) Two-photon image of a neuron in L2/3 of mouse S1 cortex, infected with a ChRmine/GCaMP6m virus. Scale bar: 10 μm. (c) Calcium signal of a neuron under different stimulation patterns at the optimal stimulation depth. (d) Changes in calcium signal caused by two-photon optogenetics at different stimulation depths under different stimulation patterns. The fitted response intensity curves are shown with solid lines. (e) Statistical analysis of axial resolutions under different stimulation schemes. Data are from 10 neurons in three mice. **p=0.0017, ratio paired t test.

Fig. 6. All-optical physiology test with different stimulation patterns on multiple neurons simultaneously in vivo. (a) Left: typical two-photon image of neurons in L2/3 of mouse S1 cortex, infected with a ChRmine/GCaMP6m virus. Locations of gray spots are the center of two excitation patterns. Right: distribution of excitation patterns to stimulate target neurons. D, disk pattern; BR, beaded-ring pattern. Scale bar: 10 μm. (b) Mean axial resolutions of calcium signals of multiple neurons under different stimulation schemes. Data are from eight groups, and each group has two neurons to be stimulated. **p=0.0086, ratio paired t test. (c) Mean peak responses of multiple neurons at their optimal stimulation depths under different stimulation schemes. Data are from eight groups, and each group has two neurons to be stimulated. ns, not significant; p=0.0638, ratio paired t test.

Fig. 7. Calcium signal of neurons with different types of stimulation patterns, at different stimulation powers. BR, beaded-ring pattern; D, disk pattern; ns, not significant. p=0.30, 0.35, 0.43, 0.12, and 0.36 for stimulation power with 3, 5, 7, 9, and 11 mW, respectively, ratio paired t test. Duration of each stimulation: 100 ms. Data are from three neurons, and each neuron is stimulated with two stimulation patterns at different stimulation powers.