Calibration of the automated z-axis of a microscope using focus functions

Applications in automated microscopy and three-dimensional microscopy require careful calibration of the microscope system. This paper presents methods for calibration of the motorized z -axis (focus or optical axis) of an automated microscope. Apart from the automated microscope the procedures require a CCD camera and a test slide containing a simple bar pattern. The calibration embraces the following characteristics of the z -axis: (a) measuring the motor step size in nanometres; (b) measuring the mechani-cal backlash in the focus mechanism of the microscope and (c) measuring the reproducibility and the stability of the focus position over time. The measurements employ focus functions to determine the z -position of the microscope stage.