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Single Cell Imaging of PI3K Activity and Glucose Transporter Insertion Into the Plasma Membrane by Dual Color Evanescent Wave Microscopy

Many signaling events involve the translocation of signaling molecules to or from the plasma membrane; however, suitable techniques to quantify the temporal relationships between such signaling events are lacking. Here, we describe an evanescent wave microscopy technique that allows parallel measurement of the recruitment and dissociation of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) labeled proteins to and from the plasma membrane in individual living cells. The selective excitation of fluorescence in a zone less than 100 nm above a cover glass enables selective imaging within the plasma membrane of adherent cells, with markedly improved resolution, lower background, and minimal phototoxicity compared to confocal microscopy and other microscopy-based assays. In the microscope design we have developed, the beams from helium-cadmium (442 nm) and argon (514 nm) lasers are merged and focused through a dove prism at an angle that yields total internal reflection. In this configuration, evanescent wave-excited fluorescence at the glass-water interface can be detected with either high or low magnification, to allow for high-resolution imaging or the study of many cells in parallel. We applied this technique to make parallel measurements of the time-course of insulin-triggered activation of phosphatidylinositol 3-kinase (PI3K) and GLUT4 glucose transporter insertion into the plasma membrane of individual differentiated 3T3L1 adipocytes using a phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]-binding pleckstrin homology domain fused to CFP, and GLUT4 conjugated to YFP. The technique should have wide applicability to various cell types and diverse signaling processes.