In vivo two-photon time-lapse imaging of filopodium formation and retraction of adult-born neurons in the OB. (A) Experimental scheme of in vivo two-photon time-lapse imaging of microglia–spine interaction. (B) Representative two-photon image of tdTomato⁺ dendrites of granule cells (red) and EGFP⁺ microglia (green) in the EPL of the adult OB. Boxed areas are enlarged in Fig. 5, C–E. Also see Video 4. (C and D) In vivo two-photon imaging of dendritic spines of adult-born periglomerular cells and granule cells. Representative z-stack two-photon images (C) and their 3D reconstruction (D) of tdTomato⁺ periglomerular (yellow arrowheads) and granule (red arrowheads) cells in Dcx-CreER^T2; R26-EPT; R26-tdTomato (EPT) mouse at 28 dpi are shown. (E) Experimental scheme of in vivo two-photon time-lapse imaging of filopodium formation and retraction. (F and G) Representative in vivo time-lapse images of tdTomato⁺ filopodium formation (F, yellow arrows) and retraction (G, pink arrows) in the EPL (n = 3 mice, pooled from three independent experiments). Asterisks (F and G) indicate spines stably observed during the whole imaging period. Numbers indicate the time in minutes from the initial image session. (H) Frequency of filopodium formation and retraction in the adult EPT and Dcx-CreER^T2; R26-D89E; R26-tdTomato (D89E) mice at 28 dpi (n = 3 mice each, pooled from three independent experiments). GL, glomerular layer; EPL, external plexiform layer; GCL, granule cell layer. Scale bars: B, F, and G, 2 µm; C, 20 µm. Data shown are mean ± SEM.