Simultaneous two-photon imaging and two-photon optogenetics of cortical circuits in three dimensions
- Columbia University, United States
Figures
Figure 1 with 4 supplements
Two-photon imaging and photostimulation microscope.
(A) Dual two-photon excitation microscope setup. HWP, half-wave plate; ZB, zeroth-order beam block; SLM, spatial light modulator; ETL, electrically tunable lens; PMT, photomultiplier tube. (B) …
Figure 1—figure supplement 1
System characterization of the spatial light modulator (SLM) in the 3D microscope.
(A) Measured point spread function (PSF) in the axial (z) direction for two-photon excitation. The FWHM is 14.5 μm, corresponding to an NA ~ 0.35. (B) Measured axial profile of a two-photon …
Figure 1—figure supplement 2
Characterization and spatial resolution of photostimulation.
(A–B) Latency (A) and jitter (B) of target pyramidal cells in layer 2/3 of mouse V1 evoked by photostimulation with different spiral duration and average laser power (3 cells in each condition; mice …
Figure 1—figure supplement 3
Cross talk from imaging into photostimulation.
Activities of neurons in layer 2/3 of mice V1 were recorded by cell-attached electrophysiology while the whole field was being scanned by the imaging laser (940 nm) at an FOV of 240 × 240 μm2 at …
Figure 1—figure supplement 4
Cross talk from photostimulation into imaging.
This example represents one of the worst cross talk situation: the bright GCaMP6s signal, the relatively strong photostimulation power (60 mW) and its long duration (2.8 s) render a strong …
Figure 2 with 1 supplement
Comparison between spiral scan and scanless holographic approaches for photostimulation.
In the scanning approach, the laser spot is spirally scanned over the cell body; in the scanless approach, a disk pattern (~12 μm in diameter) is generated by the SLM, covering the entire cell body …
Figure 2—figure supplement 1
Comparison between spiral scan and scanless holographic approaches for photostimulation.
In the scanning approach, the laser spot is spirally scanned over the cell body; in the scanless approach, a disk pattern (~12 μm in diameter) is generated by the SLM, covering the entire cell body …
Figure 3 with 1 supplement
Simultaneous holographic photostimulation of pyramidal cells in vivo.
(A) Contour maps showing the spatial location of the cells in three individual planes in mouse V1 (145 μm, 195 μm, and 245 μm from pial surface). Cells with shaded color are the targeted cells. (B) …
Figure 3—figure supplement 1
Sequential photostimulation of individual pyramidal cells in layer 2/3 from mouse V1 in vivo.
(A) Contour maps showing the spatial location of the cells in three individual planes in mouse V1 (90 μm, 120 μm, and 150 μm from pial surface). Cells with shaded color are the targeted cells. (B) …
Figure 4 with 1 supplement
Large scale photostimulation of pyramidal cells in layer 2/3 of V1 in awake mice.
(A~C) Simultaneous photostimulation of 40 cells, 43 cells and 83 cells across four planes in mouse V1 (150 μm, 200 μm, 250 μm and 300 μm from pial surface, with an imaged FOV of 480 × 480 µm2 in …
Figure 4—figure supplement 1
Simultaneous photostimulation of 50 pyramidal cells in layer 2/3 of V1 in awake mice.
(A) Contour maps showing the spatial location of the cells across four individual planes in mouse V1 (170 μm, 220 μm, 270 μm and 320 μm from pial surface). Cells with black contour are the …
Figure 5
Selective photostimulation of SOM interneurons suppresses visual response of pyramidal cells in awake mice.
(A) Experiment paradigm where the SOM cells were photostimulated when the mouse received drifting grating visual stimulation. (B) Normalized calcium traces (ΔF/F) of representative targeted SOM …
Tables
Table 1
Expression of Zernike polynomials and Zernike coefficients in Equation (1).
https://doi.org/10.7554/eLife.32671.015| Defocus | |
|---|---|
| Zernike polynomials | |
| Zernike coefficients | |
| First-order spherical aberration | |
| Zernike polynomials | |
| Zernike coefficients | |
| Second-order spherical aberration | |
| Zernike polynomials | |
| Zernike coefficients | |
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n, refractive index of media between the objective and sample; k, the wavenumber; z, the axial shift of the focus plane in the sample; u, v, coordinates on the SLM phase mask; nsinα, the NA of the objective.
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32671.016
