Fig 1.
Optical setup of holographic projector attachment.
a: Schematic of the commercial two-photon microscope and the holographic attachment. b: Detailed schematic of the holographic projector attachment. PBS: polarizing beam splitter, M: mirror, L: lens, SLM: spatial light modulator, λ/2: half waveplate. H1: conjugate image of the SLM hologram (H0). Lenses: f1 = f4 = 30 mm; f2 = f3 = 100 mm; f5 = f6 = 500 mm. c: Photographs of the setup showing the front view of the commercial microscope (left), and the back view showing the holographic attachment (right).
Fig 2.
Software control of holographic projector.
a: Interfacing between our custom SLM software and the rest of the system including the microscope’s control software (MES), which gathers the pixel resolution. b: Custom software graphical user interface showing xyz-coordinates of uncaging spots. We use the last column to encode a Laguerre-Gaussian beam of charge “L” to align the center of the SLM with respect to the optical axis. c: Two-photon 3D image of a basal dendrite with multiple spines. Positions of uncaging sites in b are indicated. d: The corresponding 8-bit phase-only hologram for projecting the multiple uncaging spots in b.
Fig 3.
Performance of the holographic system.
a: Normalized fluorescence intensity of a focal spot as a function of number of spots, N b: Normalized uncaging power per spot as a function of number of stimulation spots. Number of foci is kept constant and superfluous foci are moved outside of the holographic FOV in transverse direction. c: Measured PSF of two-photon excitation in the axial (⚬) and transverse (●) directions for 60× and 20× objective lenses. d: Normalized power as a function of displacement along the x for 60× for FOV centered at (0, 0) (⚬), (−100, −100) μm (△) and (100, 100) μm (□). e: Normalized power as a function of displacement along the x for 20× for FOV centered at (0, 0) (⚬), (−300, −300) μm (△) and (300, 300) μm (□). Filled circles with dashed line denote power distribution after amplitude weighting for FOV centered at (0, 0). f: Normalized power as a function of displacement along the z for 60× (red markers in red area) and 20× (black markers in gray area) for FOV centered at different coordinates specified in d and e. Solid lines denote empirical Gaussian fit used for amplitude weighting. Filled circles with dashed line denote power distribution for 20× after amplitude weighting for FOV centered at (0, 0).
Fig 4.
Representative synaptic integration experiment.
a: Two-photon image of a layer 2/3 pyramidal neuron labeled with OGB-1 taken with a 60× objective. Inset shows a high-magnification flattened multi-stack image of the VOI. Scale bar: 20 μm. b: VOI displayed as an xz-image with 3 representative image planes (i: z = −5 μm; ii: z = 0; and iii: z = + 5 μm. Scale bar: 5 μm. c: Somatic EPSPs of individual uncaging events (red bar indicates time point of 2P glutamate uncaging). Scale bars: 1 mV, 40 ms. d: Uncaging responses with increasing number of simultaneous uncaging sites. Scale bars: 20 mV, 50 ms. (Inset) Magnified EPSP rise times for increasing number of uncaging sites. Scale bars: 5 mV, 5 ms. e: Representative individual uncaging-evoked EPSP (top) and Ca2+ transient (ΔF/F) (bottom) measured at spine 4 (black) and nearby dendrite (gray). Scale bars: 2 mV, 20%, 500 ms. f: Input-output plot (n = 4) of measured multisite uncaging response against the arithmetic sum of individual uncaging events. Dashed line indicates linear summation, black circles represent data from a while all other markers represent data from other experiments.
Fig 5.
Synaptic integration experiment with a larger VOI.
a: Two-photon image of a layer 2/3 pyramidal neuron labeled with OGB-1 using a 20× objective lens results in a larger FOV. Scale bar: 20 μm. b: Uncaging VOIs (1, 2 and 3) targeting specific dendrites corresponding to regions indicated in a. Scale bar: 20 μm. c: Somatic action potentials evoked when simultaneously uncaging at all sites (8-10) within corresponding uncaging VOIs in b. Scale bars: 20 mV, 50 ms. d: Uncaging VOI 4 (region indicated in a) showing a dendrite that extends 16 μm along the axial direction. Scale bar: 20 μm. e: Evoked EPSPs in VOI 4 when two sites are activated separately (red and grey traces) and simultaneously (black trace). Scale bars: 1 mV, 50 ms.