Figure 5. Dynamic intravital imaging by MB-SI-TPLSM.

(a) Rotation view of a 3D fluorescence image in a germinal center of the popliteal lymph node of a mouse after footpad immunization with chicken γ-globulin (NP-CGG). Imaging was performed 9 days after immunization. Follicular dendritic cells (FDCs) were in situ stained by anti-CD21/CD35-Fab fragments coupled to Alexa568 (red) 24 h before imaging. NP-specific GFP⁺ (green) and GFP⁻ B cells were transferred into C57BL/6 mice. The communication between antigen-carrying FDC and B cells is thought to be involved in the clonal selection process of high affinity B cells. By means of MB-SI-TPLSM we were able to reveal the dynamic nature of the contacts between FDC somata and B cells (white arrowhead) but also, due to improved resolution and contrast, interactions between fine FDC processes and B cells (white arrow). λ_exc = 800 nm, scale bar = 10 µm. In the same context, we used for the staining of FDCs anti-CD21/CD35 Fab fragments coupled to ATTO590 for a better simultaneous visualization of FDCs and B cells (b–d). Time-lapse 3D fluorescence images by MB-SI-TPLSM in 85 to 105 µm depth in the lymph node (c) revealed that CD21/CD35+ immune complexes are accumulating around the B cells and that this interaction is highly dynamic (d, e). The trajectories of the CD21/35 clusters are shown in (d), whereas their statistics concerning velocity and displacement rate are summarized in (e). The contact regions between the CD21/35 clusters and B cell are shown in yellow (f). λ_exc = 860 nm, scale bar = 10 µm, grid unit = 3.5 µm.