Fig. 2. Membrane areas with anomalously high ErbB2 homoassociation colocalize with membrane domains with high ErbB2 and comparatively low ErbB3 densities. SKBR-3 breast tumor cells were labeled with FITC-4D5 and Cy3-4D5 against ErbB2, and with unlabeled H3.90.6 anti-ErbB3 antibody followed by secondary labeling with a Cy5-tagged Fab. Images A and B show the fluorescence intensity distribution of Cy3-4D5 (ErbB2) and the Cy5-tagged secondary Fab (ErbB3), respectively. The FRET efficiency for ErbB2 homoassociation is displayed in image C. The areas inside the white polygons in image C have the highest FRET values. The same areas are marked with red polygons in images A, B, D-F. These areas have very high ErbB2 density and comparatively low ErbB3 density. The areas at the head of the arrows have the highest ErbB2 and ErbB3 densities, but ErbB2 homoassociation in these pixels is lower than inside the marked areas. Image A was thresholded, and pixels with high ErbB2 fluorescence intensity are white in image D, whereas pixels with intensity below the threshold are black. Image B was `bithresholded': two intensity values were determined, and only pixels whose intensity is between the two threshold values are white in image E, all other pixels are displayed in black. Threshold values were adjusted so that white pixels in image E have low ErbB3 intensities, but still above a certain level. Pixels with intensity values lower than the lower threshold are background pixels, and they have to be excluded from the analysis. In image F a pixel is displayed in white if the pixel is white in both image D and E. The distributions of white areas in image F correlate with pixels with high ErbB2 homoassociation in image C. Bar, 1 µm.