Total internal reflection fluorescence microscopy (TIRFM) permits imaging near-membrane dynamics by selectively exciting fluorophores close to a dielectric interface. However, neither quantifying TIRF intensities across a single image, nor between frames, nor the comparison among experiments is straightforward, because the exact shape of the axial intensity decay of the evanescent wave in the sample is generally unknown, and so is the relation between axial fluorophore distance and intensity. Stray light generated in the objective and along the microscope illumination optical path as well as cell-induced scattering modify the excitation intensity and axial confinement, making the optical sectioning space dependent. The characterization of such inhomogeneous intensity distributions, in situ, is major a challenge in modern microscopy, which seeks to gain quantitative information about the abundance, localization, orientation of near-membrane fluorophores or their interaction (quenching, FRET).