Fig. 1. Sketch layout of PIE-FCCS and its role in membrane protien interaction.

(A) Diagram illustrates the optical path of dual-color PIE-FCCS. A super-continuum, pulsed laser-generated excitation beams (488 and 561 nm) that were directed into the sample by a dichroic mirror and microscope objective. The lasers were focused to a diameter of about 250 nm on the plasma membrane of live cells. Fluorescence emission was collected by the objective, filtered, and directed to single-photon counting modules connected to a TCSPC module for data recording. (B) Epifluorescence images are shown for COS-7 cells expressing EGFR-mCH (top) and EphA2-eGFP (bottom). (C) Summary data from independent experiments collected on five different days of single-cell measurements are shown for the controls and EGFR with EphA2 (***P < 0.0004). (D to F) Representative single-cell PIE-FCCS data are shown for a monomer control (SRC), dimer control (GCN4), and EGFR with EphA2 in COS-7 cells, respectively. In each graph, the green line is the autocorrelation function (ACF) for eGFP, the red line is the ACF for mCH, and the blue line is the cross-correlation function (CCF) between both protein constructs.