In micro-domain applications, such as assembly of hybrid microelectromechanical systems, optical microscopes form a critical component of visually based microassembly systems. In order to effectively use vision feedback to guide manipulation strategies using a broad range of optical microscope configurations, accurately calibrated parametric models of optical microscopes are required. Although many calibration techniques exist for macroscopic camera-lens systems, there is a dearth of literature on calibration of parametric microscope models. Optical microscope calibration has unique characteristics that are quite different from normal camera calibration, including (1) unique calibration parameters; (2) calibration patterns that must be parallel to the image plane, and (3) restriction to single-plane calibration. In this paper, we propose a parametric microscope model and calibration algorithm specifically for optical microscopes. Our approach extends existing camera calibration techniques to include the unique parameters of optical microscopes and also allows the use of a single calibration plane perpendicular to the optical axis. The validity and accuracy of our proposed microscope calibration method are tested by experiments.