Fig. 5. Image processing protocols for isolation of the signals for EGFR activation and Shc translocation and generation of three-dimensional data sets. Schematics illustrating the immunofluorescence staining are included in the leftmost panels. The image processing protocol for the analysis of receptor activation only retains the signals colocalizing with the microspheres (colored pixels in the top center panel). The positions of the microspheres are outlined in red on one slice of the three-dimensional stack of confocal images. For Shc the signal at the microspheres as well as the Shc signal in the local environment are retained. The microspheres are again designated by red pixels. To illustrate the heterogeneous subcellular distribution of Shc more clearly an unsegmented enlargement is included in the bottom center panel. The colored ring around the microsphere-associated signal represents the local environment. An xz-projection through the image stacks is included for the enlarged areas. The final three-dimensional data set consists of (1) the level of EGFR activation, (2) the Shc signal in the local environment of a microsphere, and (3) the Shc signal colocalizing with the microsphere. The pseudocolor look-up table was chosen to better illustrate which data were isolated by the image processing procedure.