Co-localization and FRET imaging between Cy3-labeled SNAP23 and Cy5-labeled GLUT1.

<p>Confocal images of Cy3-labeled SNAP23 and Cy5-labeled GLUT1 were examined by laser scanning confocal microscopy. Co-localization of Cy3-SNAP23 (green) with Cy5-GLUT1 (red) revealed yellow-to-orange areas where both probes overlapped (A). The extent of co-localization was shown in a pixel fluorogram (B). FRET efficiency maps were generated from the following images: donor emission image of Cy3-SNAP23 co-labeled with the acceptor Cy5-GLUT1 before photobleaching (C); donor emission image of Cy3-SNAP23 co-labeled with Cy5-GLUT1 after acceptor photobleaching (D); donor emission image of Cy3-SNAP23 after photobleaching overlaid with a pseudo-colored FRET image (E); and acceptor emission image of Cy5-GLUT1 after photobleaching (F). Cells were imaged and FRET efficiency images generated as described in the Method section. The FRET overlay was pseudo-colored to visualize regions of higher and lower FRET as shown by the inset color scale (E).