Multiphoton Imaging of Synaptic Structure

For synaptic structural imaging, anesthetized mice with sparsely labeled neurons within the mapped visual cortex were imaged using a Sutter MOM multiphoton microscope. The Ti: sapphire laser (MaiTai HP: Newport SpectraPhysics; 915 nm) was routed to the microscope using table optics. The power was adjusted using a rotating half-wave plate and a polarizing beam splitter. A pair of galvanometric mirrors scan the laser beams to the back aperture of the objective (Nikon 16 × 0.8 NA). The output power from the objective was set to 40-50mW. Emission signal was collected through the same objective, passed through a short pass filter to block infrared wavelengths, and routed to three GaASP PMTs after passing through appropriate bandpass filters (488/50, 540/50, and 617/73 for Teal, YFP, and TdTomato fluorescence, respectively). Image acquisition was controlled by Scanimage (Vidrio Technologies), and images were obtained at 0.16Hz. The imaging field covered 133 × 133x~150 μm (1024 × 1024 XY pixels, Z step - 1 μm). For GCaMP6 imaging, neurons within the mapped visual cortex (~100–150 μm below the dura) were imaged at 4.22 Hz in head-restrained awake mice restrained in a body tube. The excitation wavelength was set to 940 nm, and the power was adjusted (20-40mW) to avoid signal saturation. The imaging field was a single Z frame of 336 × 336 μm (256 × 256 pixels) consisting of ~50–100 cells.