Fig. 1.

Experimental and imaging setup. (a) Experimental timeline. (b) Low magnification image showing GCaMP expression throughout a coronal histological slice of an AAV-PHP.eB-injected mouse (left). Higher magnification images (right) showing GCaMP-expressing neurons in the primary visual and retrosplenial cortex, outlined by the white boxes (scale bars: 1 mm and 100 μm for low and high magnification images, respectively). (c) Green epifluorescence and reflectance images are acquired at 150 Hz using a CCD camera configured with a tandem lens optical setup and bandpass emission filter. (d) Blue and green LEDs are sequentially illuminated at the beginning of each frame’s 6.67-ms exposure period for a duration of 5.5 ms to produce fluorescence and reflectance images each at 75 Hz. (e) Example transcranial images of green fluorescence (evoked by the blue LED) and green reflectance (evoked by the green LED) from a PHP.eB-GCaMP6s mouse (arrows: anterior/posterior and lateral/medial; asterisk: bregma; $\text{scale bar}=2\mathrm{mm}$). (f) Example trial-averaged visually evoked responses measured from contralateral V1 (see Sec. 2) for Thy1-GFP mice (top) and tetO-GCaMP6s mice (bottom). Blue LED-evoked epifluorescence is shown in blue, and green reflectance is shown in green. Magenta lines demonstrate 5-ms flash stimulus. Blood artifact-corrected responses for each mouse (right, shown in black) are represented by the subtraction of reflectance signal (green) from the fluorescence signal (blue).