Figure 2.

Visualization of cognate antigen capture from FDC by two-photon microscopy. (A) Two-photon microscopy of a follicle within an explanted LN from a mouse that had been HEL-PE (red) immunized 36 h earlier and had received CFSE⁺ MD4 B cells (green) and CFP tg polyclonal B cells (cyan) 12 h before. The capsule is visible in blue by second harmonic emission from collagen. (B) Enlarged view of boxed region in A highlighting several beaded HEL-PE⁺ processes (arrowheads). (C) Time-lapse images of a CFSE⁺ MD4 B cells encountering a HEL-PE⁺ process within the follicle, capturing and moving away with a large aggregate of HEL-PE (arrowheads), as presented in Video 2. Elapsed time is shown as minutes:seconds. Data in A–C are representative of three experiments with five mice. (D) Frequency of CFSE⁺ MD4 B cells or CFP tg noncognate B cells contacting HEL-PE⁺ FDC processes for the indicated amounts of time (202 contacts of 127 MD4 B cells, 277 contacts of 81 CFP B cells). Dark shading indicates that the contact was observed for its entirety during the imaging period; light shading indicates that the contact had formed before imaging began or persisted after the end of imaging. (E) Velocities of CFSE⁺ MD4 B cells or CFP tg noncognate B cells that were contacting HEL-PE⁺ FDC processes (in contact, dark shading) or were not contacting such processes (not in contact, light shading). Median velocities are indicated by numbers and arrowheads. (F) Contacts between CFSE⁺ MD4 B cells and HEL-PE⁺ FDC processes that resulted in antigen capture (Ag capture +) or were not accompanied with detectable antigen capture (Ag capture −). Median contact durations (bars) are 6.5 min for contacts with capture (n = 19) and 3.0 min for contacts without capture (n = 183). Bars: (A) 50 µm; (B) 20 µm; (C) 10 µm. Data in D, E, and F are pooled from three experiments.