A spatially resolved total internal reflection fluorescence correlation microscopy (TIR-FCM) system is constructed with an electron multiplying charge-coupled device (EMCCD) camera. The system was used to determine diffusion coefficients of lipid molecules in a planar lipid bilayer, and lipids and epidermal growth factor receptor (EGFR) proteins on cell membranes of Chinese Hamster Ovary (CHO) cells. The evaluation of the "cross talk" between neighboring pixels suggests that a higher degree of multiplexing can be achieved than was previously proposed [Kannan, B. et al. Anal. Chem. 2006, 78, 3444-51] using the same camera with a focused laser excitation. The best time resolution possible with this system is 4 ms for a region of interest comprising 20 lines in the CCD and is good enough to determine membrane diffusion in lipid bilayers and of membrane proteins in living cells. In this work, using a TIR-FCM setup, 1600 autocorrelation functions were measured simultaneously with a time resolution of 4.8 ms. This area corresponds to a 40 x 40 pixel region of interest with a dimension of 11.3 x 11.3 microm2 and is sufficiently large to allow the measurement of the lower membrane of a whole cell simultaneously.
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