In vivo two-photon calcium imaging dataset_Day 1

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.本研究采用共振扫描式双光子显微镜（B-scope；Thorlabs，美国）与调谐至920 nm的钛蓝宝石脉冲激光器（Chameleon Ultra II，Coherent，美国）开展双光子钙成像实验。实验中，通过带通滤波器（525/50，Semrock，美国）分离GCaMP6f荧光信号，并由GaAsP光电倍增管（Hamamatsu，德国）完成信号探测。成像过程通过20倍水浸物镜（数值孔径1.00；Olympus，日本）采集，采用双向扫描模式，分辨率为256×256像素（视场300×300 µm），帧率为14.7 Hz（经4次实时平均），成像操作由ThorImageLS成像软件（2.4版本）控制。为避免虚拟现实（VR，Virtual Reality）显示屏的光线泄漏进入显微镜系统，我们在显微镜物镜与头部固定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。每只动物均选取皮层深度120~180 µm处的单个2/3层皮层焦平面进行成像，并在多日内对同一压后皮层（RSC，retrosplenial cortex）区域开展重复成像。