In a linear tomosynthesis scanner designed for imaging histologic samples of several centimeters size at 10 µm resolution, the mechanical instability of the scanning stage (±10 µm) exceeded the resolution of the image system, making it necessary to determine the trajectory of the stage for each scan to avoid blurring and artifacts in the images that would arise from the errors in the geometric information used in 3D reconstruction. We present a method for online calibration by attaching a layer of randomly dispersed micro glass beads or calcium particles to the bottom of the sample stage. The method was based on a parametric representation of the rigid body motion of the sample stage-marker layer assembly. The marker layer was easy to produce and proven effective in the calibration procedure.