Retrieving the axial position of fluorescent light emitting spots by shearing interferometry

A method for the depth-resolved detection of fluorescent radiation based on imaging of an interference pattern of two intersecting beams and shearing interferometry is presented. The illumination setup provides the local addressing of the excitation of fluorescence and a coarse confinement of the excitation volume in axial and lateral directions. The reconstruction of the depth relies on the measurement of the phase of the fluorescent wave fronts. Their curvature is directly related to the distance of a source to the focus of the imaging system. Access to the phase information is enabled by a lateral shearing interferometer based on a Michelson setup. This allows the evaluation of interference signals even for spatially and temporally incoherent light such as emitted by fluorophors. An analytical signal model is presented and the relations for obtaining the depth information are derived. Measurements of reference samples with different concentrations and spatial distributions of fluorophors and scatterers prove the experimental feasibility of the method. In a setup optimized for flexibility and operating in the visible range, sufficiently large interference signals are recorded for scatterers placed in depths in the range of hundred micrometers below the surface in a material with scattering properties comparable to dental enamel.