Figure 1.

Comparison between epi-fluorescence and TIRF imaging. (A) Epi-fluorescence image of two GFP-tagged MHC-I fibroblasts. The nuclei (dark) are visible and patches of fluorescence are not well localized. (B) The same two cells, in the same focal plane, imaged using TIRFM. Cells appear two-dimensional, the membrane contour is sharp, and the MHC-I clusters are easily distinguished (see zoomed-in inset). (Circle, inset) Example of selection of a clearly distinguishable cluster. (C) A time-dependent plot of the average intensity in an area as was indicated (see circle in panel B). In this particular case, a new delivery is observed (indicated by an arrow). The intensity decays exponentially with time both before and after the delivery. The data after the delivery is fitted with an exponential decay (solid line). (D) Intensity profiles as a function of position of the cluster analyzed in panel C, at 10 different time points (see waterfall plot). The profiles are laterally displaced along the x axis for clarity, to form a pseudo-three-dimensional view of the spatial evolution of a single cluster with time; there is no lateral motion of the cluster peak in reality. (Arrow) Time progresses from back to front. The cluster intensity and dimensions are seen to change with time: the cluster is bright and narrow upon delivery, becoming dimmer and wider as time progresses.