Abstract
Total internal reflection fluorescence (TIRF) microscopy is a commonly used method for studying fluorescently labeled molecules in close proximity to a surface. Usually, the TIRF axial excitation profile is assumed to be single-exponential with a characteristic penetration depth, governed by the incident angle of the excitation laser beam towards the optical axis. However, in practice, the excitation profile does not only comprise the theoretically predicted single-exponential evanescent field, but also an additional non-evanescent contribution, supposedly caused by scattering within the optical path or optical aberrations. We developed a calibration slide to directly characterize the TIRF excitation field. Our slide features ten height steps ranging from 25 to 550 nanometers, fabricated from a polymer with a refractive index matching that of water. Fluorophores in aqueous solution above the polymer step layers sample the excitation profile at different heights. The obtained excitation profiles confirm the theoretically predicted exponential decay over increasing step heights as well as the presence of a non-evanescent contribution.
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