After a minimum of one week after surgery wide-field imaging was performed at 15 Hz using a 470 nm LED (M470L3, Thorlabs) to illuminate the area. Imaging was performed using an ORCA-Flash 4.0 V3 (Hamamatsu) camera and a 4x microscope objective (4x Nikon Plan Fluorite Imaging Objective, 0.13 NA, 17.2 mm WD). Excitation light passed through an aspheric condenser lens (ACL2520U-DG15, Thorlabs), a filter (ET470/40, Chroma) and was reflected into the light path by a 495 nm longpass dichroic (FF495-Di03-25x36, Semrock) to reach the brain. Emitted light passed through the same 495 nm longpass dichroic as well as a 749 nm shortpass dichroic (FF749SDi01-25x36x3, Semrock,) and an emission filter (HQ525/50, Chroma) before reaching the camera. Spontaneous activity of awake mice running freely on a treadmill was acquired for 2-4 minutes to exclude the occurrence of aberrant activity in transgenic mice60 (link). Calcium signal time series were obtained from the average pixel intensity.
To locate the barrels in the FOV single whiskers were threaded into a glass capillary and deflected 10-30× for 1 s with a piezo oscillating at 10 Hz (sinusoidal) every 10 s whilst performing wide-field imaging as described above. To locate the barrel centers, we calculated event-triggered averages of the movies by aligning the frames to the start of the whisker deflections using custom-written Python scripts.
To locate the barrels in the FOV single whiskers were threaded into a glass capillary and deflected 10-30× for 1 s with a piezo oscillating at 10 Hz (sinusoidal) every 10 s whilst performing wide-field imaging as described above. To locate the barrel centers, we calculated event-triggered averages of the movies by aligning the frames to the start of the whisker deflections using custom-written Python scripts.