Measuring incidence angle for through-the-objective total internal reflection fluorescence microscopy

Total internal reflection fluorescence (TIRF) microscopy has the exciting laser beam incident beyond critical angle from the glass side of a glass/aqueous interface formed by the coverslip and aqueous sample. The aqueous side evanescent field decays exponentially with distance from the interface with penetration depth depending on incidence angle. Through-the-objective TIRF has the exciting laser focused at the back focal plane (BFP) creating a refracted parallel beam approaching the interface in the small gap between objective and coverslip, making incidence angle challenging to measure. Objective axial scanning does not affect incidence angle but translates beam and interface intersection detected by the fluorescence center of mass from fluorescent spheres attached to the aqueous side of the interface. Center of mass translation divided by the axial translation is the tangent of the incidence angle that is sampled repeatedly over objective trajectory to obtain a best estimate. Incidence angle is measured for progressively larger radial positions of the focused beam on the BFP. A through-the-objective TIRF microscope, utilizing a micrometer and relay lenses to position the focused beam at the BFP, is calibrated for incidence angle. Calibration depends on microscope characteristics and TIRF objective and is applicable to any interface or sample.