In vivo two-photon calcium imaging dataset_Day 4
Two-photon calcium imaging was performed using resonant scanning two-photon microscopy (B-scope; Thorlabs, USA), and a Ti:Sapphire pulsing laser (Chameleon Ultra II, Coherent, USA) tuned to 920 nm. GCaMP6f fluorescence emission was isolated by a band-pass filter (525/50, Semrock, USA) and detected by a GaAsP photomultiplier tube (Hamamatsu, Germany). Images were acquired through a 20x water immersion objective (1.00 N.A.; Olympus, Japan) with a frame rate of 14.7 Hz (real-time averaging by 4) for bidirectional scanning at a resolution of 256 x 256 pixels (300 x 300 µm field of view) and controlled by ThorImageLS imaging software (version 2.4). In order to prevent light leakage from the VR displays into the microscope, a custom-made black foam ring was used between the microscope objective and the head-plate. Imaging and behavioral data were synchronized by custom-written code (MATLAB, MathWorks, MA, USA). Images were collected at a single L2/3 focal plane per animal at cortical depths between 120 and 180 µm, and the same RSC region was imaged across multiple days.