Sutter movable objective microscope

2PCI was performed on a custom microscope setup which included a Sutter movable objective microscope (Sutter Instruments, Novato, CA) with a resonant scanner (Cambridge Instruments, Bedford, MA). Data acquisition was controlled by a customized version of Scanbox (Neurolabware, Los Angeles, CA). GCaMP6s was excited by a Ti:sapphire laser (Chameleon Ultra II, Coherent, Santa Clara, CA) at 920 nm. Imaging was collected at 1 or 3 planes. For uniplanar experiments, continuous unidirectional scanning was done at 15.49 Hz. For multi-planar experiments, an optotuned lens was used to alternate between depths, and a scanning rate of 5.16 Hz per plane was used. Planes were set ~20–30 μm apart. An area of approximately 500 × 720 μm (some experiments 800 × 1230 μm or 570 × 870 μm) was imaged using a 16x, 0.8 NA objective lens (Nikon Corporation, Tokyo, Japan) through the headframe filled with Immersol-W (Carl Zeiss Microscopy). Prior to imaging, mice were acclimated to the running wheel and visual stimulus setup over 3 days of training sessions. Running speed was recorded using a rotary encoder. During at least one of these training sessions, GCaMP6s expression was checked in V1. If the imaging field of view (FOV) over the area of expression was obscured due to tissue growth or had poor expression of GCaMP6s, that region was not imaged. For each mouse, we imaged 1 FOV per session, per day.