New Turf for CFP/YFP FRET Imaging of Membrane Signaling Molecules Jenafer Evans, David T Yue  Neuron  Volume 38, Issue 2, Pages 145-147 (April 2003) DOI: 10.1016/S0896-6273(03)00234-4

Figure 1 TIRF Microscopy Enables Selective Detection of FRET from Fluorophore-Tagged, Membrane-Associated Molecules Laser excitation (454 nm) propagates through the immersion oil and coverslip at the critical angle and strikes the coverslip/specimen interface where there is a change in refractive index. This configuration results in total internal reflection of the incident light, but an evanescent field propagates through a thin segment of the specimen within 100 Å of the coverslip. This allows selective excitation of the CFP-tagged channels in this segment, while CFP-tagged channels in deeper regions of the cell remain dormant. FRET in the membrane segment will result in fluorescence emission from YFP-tagged channel subunits, and such YFP fluorescence is selectively captured in the microscope output. This imaging strategy eliminates confounding FRET signals from channels trapped in intracellular compartments. Neuron 2003 38, 145-147DOI: (10.1016/S0896-6273(03)00234-4)