Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy

Abstract

Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. Here, we developed a custom three-photon microscope optimized to image a vertical column of the cerebral cortex > 1 mm in depth in awake mice with low (<20 mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free three-photon imaging. We image functional visual responses of neurons expressing GCaMP6s across all layers of the primary visual cortex (V1) and in the subplate. These recordings reveal diverse visual selectivity in deep layers: layer 5 neurons are more broadly tuned to visual stimuli, whereas mean orientation selectivity of layer 6 neurons is slightly sharper, compared to neurons in other layers. Subplate neurons, located in the white matter below cortical layer 6 and characterized here for the first time, show low visual responsivity and broad orientation selectivity.

Fig. 1

Schematic of upright three-photon microscope and layout of the modeled objective lens. a Femtosecond laser pulses from the pump laser (1045 nm) were used to pump the noncollinear optical parametric amplifier (NOPA) to obtain 1300 nm excitation wavelength. The pulse width was elongated using a two-prism pair system and the repetition rate was doubled to 800 kHz by adding a delay line. Power control of laser pulses was performed using a combination of a half-wave plate (HWP) and polarizing cube beam splitter (PCBS). A quarter-wave plate (QWP) was used to control the polarization state of the laser pulses to maximize third harmonic generation (THG) signal on the mouse brain. Laser beams were scanned by a pair of galvanometric scanning mirrors (SM), which were then imaged on the back aperture of a 1.05 NA, 25× objective lens (OL) by a pair of a scan lenses (SL) and a tube lens (TL). Mice were placed on a two-axis motorized stage. The objective was placed on a one-axis motorized stage. Emitted light was collected by a dichroic mirror (CM1, FF705-Di01,Semrock; CM2, Di02-R488, Semrock; CM3, FF555-Di03, Semrock), collection optics (CO), laser blocking filters (BF, FF01-670/SP), and nonlinear imaging filters (FA, FF01-433/24-25, Semrock; FB, FF03-525/50, Semrock; FC, FF01-630/92) and corresponding collection optics (COA, COB, and COC) for each photomultiplier tube (PMT A, PMT B, and PMT C). To make laser ablation marks at different depths of cortex, the output of the pump laser (1045 nm) was sent to the microscope using a mechanical shutter (MS) and a long pass dichroic mirror (DM, Di02-R1064, Semrock). b The optical layout of the objective modeled in Zemax at 1300 nm. The clear aperture of the objective was 15 mm and the working distance was 2.05 mm while using seawater as a sample. c Huygens point spread function results for 4 different scanning angles (0°, 0.5°, 1°, and 2°) on the objective. Numerical apertures in all cases were 1.02 and Strehl ratio values decreased from 0.997 to 0.979 while increasing the scanning angle

Fig. 2

Characterization of optical properties of live mouse cortex at 1300 nm excitation wavelength. a Determining extinction length via tissue ablation. Percent of damage ranges from 0 to 100% with respect to laser energy on the tissue surface. Threshold energy (E_th) is the energy which results in 50% damage. For ablation at 150 µm depth, E_th is 25.7 nJ. b Representative images before ablation (BA) and after ablation (AA) for three pulse energies (10, 25 and 50 nJ) at 150 µm depth. Arrows show the location of target region for the ablation before and after applying laser pulses for each pulse energy. c Semi-logarithmic plot of threshold energies for 4 different depths results in extinction length (L_ext) of 270.3 ± 12.7 µm and threshold fluence (F_th) of 1.04 ± 0.07 J/cm². d Semi-logarithmic plot for ratio of PMT signal (S) and cube of laser power (P) with respect to imaging depth for third harmonic generation (THG) imaging. Slope of this curve results in 242 ± 40 µm extinction length for the cortex (L_ext,cortex), and 107 ± 13 µm extinction length for the white matter (L_ext,WM). Laser power was increased by two-fold every 150 µm in depth. Scale bar in (b) represents 100 µm. Values shown are mean ± s.e.m

Fig. 3

Characterization of visual responses of different layers in V1 of awake mice. a Three-dimensional rendering of a sequence of 450 lateral three-photon images acquired with 2-µm increment. Green color represents GCaMP6s signal, and magenta color represents label-free THG signal generated in the blood vessels in the visual cortex and myelin fibers in the white matter. Scale bar, 100 µm. b Selection of lateral images from layer 2/3, 4, 5, and 6. Scale bar, 50 µm. Field of view in all lateral images is 250 μm. c Average calcium responses (ΔF/F) for representative cells in each layer over 10 trials in response to oriented gratings moving in specific directions (arrows above each trace) and their orientation tuning curves in polar plots. Discontinuities in (ΔF/F) traces are due to the randomization of each stimulus direction in each trial. (ΔF/F) scale bars correspond to 100% and time scale bar corresponds to 10 s as shown in the bottom right panel

Fig. 4

Population neuronal responses in each layer across 5 animals. Global orientation selectivity index (gOSI), local orientation selectivity index (OSI), and directional selectivity index (DSI) of neurons in layers 2/3, 4, 5, and 6. Total numbers of orientation selective neurons imaged in each animal are 191, 181, 176, 171, and 155. Their laminar location is listed in Supplementary Table 3

Fig. 5

Characterization of visual responses of subplate neurons in the white matter below V1 layer 6. a Schematic coronal section of V1 showing locations of cortical (blue) and subplate (green) neurons surrounded by bundles of myelinated axons (red). b Left: Three-photon image of subplate neurons (green) and myelinated axons (magenta) in the white matter. Scale bar, 50 µm. Right: Average calcium responses (ΔF/F) for representative cells in response to oriented gratings drifting in specific directions (arrows above each trace) and their orientation tuning curves in polar plots. Discontinuities in (ΔF/F) traces are due to the randomization of each stimulus direction in each trial. c Population responses of subplate neurons showing global OSI, local OSI, and DSI. Total numbers of subplate neurons imaged in each animal are 43, 48, 38, 35, and 41