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In the context of virtualized reality based telemicromanipulation, we present in this paper a visual calibration technique for optical microscope coupled with a CCD camera. In contrast to previous approaches, a virtual calibration pattern is constructed using the micromanipulator with a sub-pixel localization in the image. We also present a new camera calibration algorithm based on Parallel Single-Plane properties. The proposed procedure leads to a linear system from which the solution gives directly both intrinsic and extrinsic parameters of the geometrical model. Both computer simulation and real data have been used to test the proposed technique, and very good results have been obtained. Compared with classical techniques, our method provides an alternative technical solution, easy to use and flexible in the context of micromanipulation and virtual reality.