The invention relates to a method for calibrating a deflection unit in a TIRF microscope, by means of which an angle of incidence of excitation light onto a specimen is adjusted, wherein a setting of said deflection unit is adjusted such that the pertaining angle of incidence is definitely greater or definitely smaller than an anticipated critical angle for total reflection of the excitation light on a surface of a used specimen, the angle of incidence is scanned by varying the setting of said deflection unit in the direction of an anticipated critical angle, an intensity of an optical response of the used specimen elicited by the excitation light being measured for each setting of the deflection unit, the intensity of the optical response of the specimen used is measured at least for a number of settings of the deflection unit until the intensity of the optical response of the specimen used traverses a flank, and the setting of the deflection unit pertaining to the flank is stored as the setting for the critical angle for total reflection at the specimen used.