Incidence angle calibration for prismless total internal reflection fluorescence microscopy.

We propose a calibration routine useful to evaluate the incident angle in total internal reflection fluorescence (TIRF) microscopy. This procedure is based on critical angle measurements conducted in the back focal plane (BFP) of the objective. Such BFP imaging can be easily implemented on any TIRF setup, making this technique very attractive. Calibration exactitude was demonstrated by comparing the theoretical angular dependence of the electric field intensity |E|