Time-lapse FRET imaging using TIRFM reveals the spatial and temporal dynamics of the change in the lipid environment of the BCR during antigen contact. (A) Time-lapse TIRFM images of CH27 B cells expressing Igα-YFP and Lyn16-CFP contacting a planar lipid bilayer containing only ICAM-1 or ICAM-1 and the antigen PC₁₀-BSA. Three-channel TIRFM images were acquired using an electron-multiplier CCD camera. The CFP and YFP images are shown. FRET was calculated by sensitized acceptor emission as described in Materials and methods. Corrected net FRET (Fc = F − β × D − γ × A) and FRET efficiency from representative cells are shown as color-coded scaled images. A merged image of CFP (red), YFP (blue), and Ea (green) is also shown, and the relative FIs across the cells indicated by red lines (scale, 20 μm) are given. (B) FIs of Igα-YFP, Lyn16-CFP, and FRET efficiencies with time for the cells imaged in A. Note that a 10:1 ratio of CFP to YFP FIs is equivalent to a 1:1 molar ratio of CFP to YFP under our experimental setting as described in Materials and methods. (C) Mean + SEM (error bars) of the FRET efficiencies for multiple cells expressing Igα-YFP and either Lyn16-CFP (12 cells) or Ger-CFP (six cells). The data are from one of three independent experiments.