Simulated P/S and P+2S as a function of spherical indentation depth z_d. The geometry of the indentation structure is schematically shown at the bottom. The individual P and S intensities are also shown. The exact Q_┴ versus z and Q_‖ versus z collection efficiencies, as calculated from Hellen and Axelrod (1987) for NA = 1.49, were used to generate these curves. Parameters assumed in the simulation are R = 150 nm (solid) or 250 nm (dashed) radius spherical indentation, diI labeling in both the indentation and planar areas, convolution with a point-spread function of half-width 211 nm to simulate the optical resolution limit, an evanescent field e⁻¹ depth of 110 nm, an angle β between diI dipoles and the normal to the plane of the membrane of 69° (see Test of theory…), and a distance between the TIR substrate and the planar parts of the structure of 50 nm. Images generated with these parameters are then pixelated in simulation with a pixel size of 73 nm. Only those pixels whose centers fall with a radius of 4-pixel widths (292 nm) from the center of the image are counted toward total intensities P and S. The values for P and S that would be expected from a completely flat membrane are seen as the ordinate intercepts at z_d/R = 0. The curves are changed only slightly if the collection efficiencies Q_┴ and Q_‖ are equal at all z positions.