Fig. 6

Time-lapse SIM of phagocytosis in cultured microglia. Cultured microglia were treated for 18 h with LPS to induce activation or with vehicle as control (CTRL) and exposed to fluorescent 100 nm beads. SIM started 20 min after bead exposure and was repeated with a 30-min time frame (a). Representative SIM images showing cultured unstimulated microglia (CTRL, b) or LPS-activated microglia (LPS, b′) over the time points. Scale bars = 2 μm. Beads were uptaken only by LPS-activated microglia as confirmed by quantification of the number of internalized beads (c). GLCM analysis showed time-dependent decrease of angular second moment (ASM) and inverse difference moment (IDM, d, e) in phagocytic microglia, indicative of decreased pixel homogeneity. Conversely, entropy increased over time with phagocytosis (e). Mean ± sd, n = 9. Two-way ANOVA for repeated measures (treatment effect had p < 0.0001 in c, e, and f and p = 0.0005 in d) followed by Dunnett’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS t = 20′. Immunofluorescence on LPS microglia fixed after the final time point and SIM revealed that beads (green) were mostly uptaken by lysosomes (CD68, red, g) as further supported by the magnified field with the xz axis (g′). Scale bars = 2 μm