Extended Data Fig. 9 ∣. High-speed trans-segmental imaging of microglia calcium activity in freely moving CX3CR1-GCaMP5g-tdTomato mice.

a,c,e,g, Maximum intensity projection images from four example time-lapse recordings in a freely behaving CX3CR1-GCaMP5g-tdTomato mouse ~5 weeks after tamoxifen injection (Supplementary Fig. 1; Supplementary Video 5) overlaid with ~10 μm × 10 μm ROIs. The recordings, which show microglia calcium activity during animal rest, tail pinch, locomotion, and turning were acquired over a ~105 min period after macroscope mounting. Only active ROIs above the indicated ΔF/F thresholds are shown. The chosen ΔF/F thresholds are based on image noise levels, depend on fluorescent indicator expression, and were consistently applied across all animals of the same strain. b,d,f,h, Top, turning motion, pressure stimulus amplitude, and locomotor activity during the example recordings shown in a, c, e, and g and Supplementary Video 5. Center, all computationally identified active ROIs. Bottom, 500 representative inactive ROIs. The spatiotemporal properties of these microglial single-cell and population calcium activities are distinct from motion-induced artifacts in CX3CR1-GFP mice (Extended Data Fig. 8; Supplementary Video 2).