Conformational change in the BCR cytoplasmic domains correlates temporally and spatially with the BCR's association with the lipid raft probe. (A) J558L cells expressing B1-8γ-CFP and Igα-YFP were allowed to spread on bilayers containing NIP₁₄-BSA. TIRFM images were acquired, and FRET efficiency was calculated from tracking of individual clusters of the BCR as described in Materials and methods. Data represent mean ± SEM (error bars) from 14 clusters from multiple cells in four independent experiments. The corrected average FI of the BCR in clusters is also shown. Corresponding phases of the cluster lifetime are shown at the top. (B) Images of J558L cells expressing B1-8γ-CFP and Igα-YFP spreading on a NIP₁₄-BSA–containing bilayer. IgG-CFP (left) and YFP/CFP fluorescence ratio images at 442-nm laser illumination at the indicated times from the initial contact are shown.