Methods and systems for autofocusing of an imaging system are presented. Provided is an imaging system and an optical interferometry system for generating one or more images corresponding to a target region in a subject. The method provides calibration information that identifies a focal position of the optical interferometry system corresponding to a determined focal position of the imaging system. A subsequent focal position of the imaging system is determined for generating a desired image corresponding to at least one of another target region in the subject and another position of the target region relative to the imaging system based on the calibration information.