Probe systems and methods of characterizing optical coupling between an optical probe of a probe system and a calibration structure. The probe systems include a probe assembly that includes an optical probe, a support surface configured to support a substrate, and a signal generation and analysis assembly configured to generate an optical signal and to provide the optical signal to the optical device via the optical probe. The probe systems also include an electrically actuated positioning assembly, a calibration structure configured to receive the optical signal, and an optical detector configured to detect a signal intensity of the optical signal. The probe systems further include a controller programmed to control the probe system to generate a representation of signal intensity as a function of the relative orientation between the optical probe and the calibration structure. The methods include methods of operating the probe systems.