Spatial distribution of DC molecules in the synapse with T cells. (A) DCs were pulsed with an influenza vaccine, combined with autologous, influenza-expanded CD4⁺ T cells, incubated for 40 minutes, fixed, and labeled. Rows A through D show the contact area (indicated by arrowhead in DIC images at far right) of distinct T cells (indicated by arrows) where they are bound to a DC. T cells C and D are bound to the same DC. Panels 1 to 5: Confocal microscopy of 0.5-μm-thick sections taken in 0.25-μm steps with a C-Apochromat 63×/1.20 W Corr objective; image 1 was closest to the slide. CD54, green; HLA-DR, blue. Panels A_Z to D_Z are overlays of sections 1 to 5 and the pertinent DIC image. Arrows point to T cells (the black arrow points to a cell underneath the DC). Arrowheads show the contact site analyzed in respective panels 1 to 5. Image resolution was increased 5-fold by linear maximization of the tonal range for each color and by background reduction by equal increase of input levels of shadows for all color channels. Bars in fluorescence images represent 1 μm; bars in DIC images represent 10 μm. (B) Line-by-line fluorescence intensity measured and plotted for each fluorophore in an optical slice through frame C3, panel A (as diagrammed to the right of plots). Intensity distribution along the lines demonstrates the unorganized distribution of synapse molecules in the x-y plane.