Supplementary Figure 3: Image registration and calcium transient calculation.
From: Thalamus provides layer 4 of primary visual cortex with orientation- and direction-tuned inputs
(a) Example frames of a calcium imaging stack of thalamic boutons at 350 μm below pia. Scale bar: 10 μm. (b) Average of 3,120 frames before and after iterations of registration. (c) Area within the green square in b. Red numbers: six regions of interest (ROIs) that are putative thalamic boutons. Scale bar: 10 μm. (d) Average fluorescent signal intensity of each ROI for each frame of the registered stack (upper panels, only part of the data shown); The tallest bin of the fluorescent signal histogram (right panels) is set as the baseline fluorescence F0 (magenta dots in left panels). Calcium transient ΔF/F (lower panel) is then calculated as ΔF/F=(F – F0)/F0. ROI6: a representative fluorescent signal trace across 3,000 frames (~45-minute acquisition for orientation, spatial, and temporal frequency tuning) measured with 156 mW post-objective power.
