Figure 9.

Criteria used in the selection of optical planes from three-dimensional reconstructions of mature immunological synapses, for the illustration of SMAC formation in vivo. 90° rotations of three-dimensional reconstructions were made to observe the xz optical planes 1, 2, and 3 through axis y. Images A1, A2, and A3 show xz optical planes from image A. The immunological synaptic interface seen in optical plane xz (1) is illustrated in A1; this optical plane was selected as the one in which the T cell's nucleus could not be visualized. Optical planes 2 and 3 (illustrated in A2 and A3) show the T cell's nucleus, demonstrating that these optical planes are at a distance from the synaptic interface and thus do not allow observation of the SMAC. Note that the optical plane 1 is the only one that shows the SMAC. yz optical planes 4, 5, and 6 were obtained from a 90° rotation of image B through the x axis. In plane 5 (illustrated in A5) the TCR is closely apposed to the infected cell without any intervening LFA-1; this plane is taken through the center of the c-SMAC. In images of planes 4 and 6 (illustrated in A4 and A6) LFA-1 immunoreactivity, rather than TCR, is in close apposition of the infected cell, indicating that these planes are taken through the p-SMAC. Note that this detailed three-dimensional reconstruction analysis was performed on the synapse illustrated in Fig. 8 A.