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.双光子钙成像（two-photon calcium imaging）实验采用共振扫描双光子显微镜（resonant scanning two-photon microscopy，型号B-scope；Thorlabs，美国）及调谐至920 nm的钛蓝宝石脉冲激光器（Ti:Sapphire pulsing laser，型号Chameleon Ultra II，Coherent，美国）开展。GCaMP6f荧光信号经带通滤波器（band-pass filter，型号525/50；Semrock，美国）分离后，由GaAsP光电倍增管（GaAsP photomultiplier tube，Hamamatsu，德国）采集。图像采集采用20倍水浸物镜（20x water immersion objective，数值孔径1.00 N.A.；Olympus，日本），以14.7 Hz的帧频（经4次实时平均）进行双向扫描，图像分辨率为256×256像素（视场300×300 µm），并通过ThorImageLS成像软件（版本2.4）控制。为避免虚拟现实（virtual reality, VR）显示器的光线泄漏进入显微镜系统，在显微镜物镜与头板之间加装了定制黑色泡沫环。成像数据与行为数据通过自定义代码（MATLAB，MathWorks，美国马萨诸塞州）实现同步。每只动物均在皮层深度120~180 µm的L2/3单个焦平面采集图像，并在多日内对同一压后皮层（retrosplenial cortex, RSC）区域进行成像。