Cells were placed on planar fluid lipid bilayers lacking or containing antigens. TIRF images were acquired at 37°C on a heated stage by an Olympus IX-81 microscope supported by a TIRF port, CascadeII 512 × 512 electron-multiplying CCD camera (Roper Scientific), and an Olympus 100 × 1.45 N.A. and Zeiss 100 × 1.4 N.A. objective lens. The acquisition was controlled by Metamorph software (Molecular Devices). The exposure time was 100 ms unless specially indicated. Three types of lasers were used: a 442 nm solid state laser; a 488 nm and 514 nm argon gas laser, and a 568 nm and 647 nm red krypton and argon gas laser. When indicated, B cells were labeled with Fab anti-Ig as specified in each figure, washed twice, and placed on lipid bilayers. B cells were then fixed 10 min after being placed on antigen-containing lipid bilayers. The fixed cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS), and then incubated in blocking buffer. The cells were incubated with rabbit antibody against p-ZAP70 (pY319)-Syk (pY352), washed three times, and then incubated with Alexa 488– or Alexa 647–conjugated F(ab')2 goat antibodies specific for rabbit IgG (Invitrogen). BCR and pSyk images were acquired by two-color TIRF microscopy through multidimensional acquisition mode controlled by Metamorph software. YFP, Alexa Fluor 568, and Alexa Fluor 647 were excited by the 488 nm, 568 nm, and 647 nm lasers, respectively. YFP, Alexa Fluor 568, and Alexa Fluor 647 emission fluorescence were collected by 550 ± 40 ET BP, 605 ± 40 BP, and 665 LP emission filters through a 488 and 568 and 647 dichroic wheel filter cube. Intracellular staining for SAP97 with Alexa Fluor 647–conjugated Fab anti-SAP97 antibody was by the procedure described for the intracellular staining of pSyk. TIRF images were background subtracted with Image Pro Plus. The MFIs of BCR, SAP97, and pSyk microclusters within the contact area were measured with Image J.