Patent ID: 8908271

Claim:
Method of operating a laser scanning microscope having a manipulation scanning system and an imaging scanning system having respective scan fields defined by respective first and second illuminating lights independently controlled in the x- and y-directions, and a beam-combiner for combining the illuminating lights of the manipulation and imaging scanning systems, the manipulation scanning system being controllable to provide a stationary focus, and the imaging system having an object field, wherein the coordinates of the x- and y-directions of the scan field of the manipulation system are represented as ordered pairs (k, j) and the coordinates of the x- and y-directions of the scan field of the imaging scanning systems are represented as ordered pairs (m, n), the method comprising the steps of: creating a reference pattern of a minimum of three different points on an unstructured sample in the scan field of the manipulation scanning system through illumination-induced modification of the sample by stationary focus of the first illuminating light at a minimum of three different scan field positions; using the imaging scanning system to detect an image of the reference pattern created on the sample in the step of creating the reference pattern; creating an overlap of the reference pattern created with the manipulation scanning system with the image of the reference pattern detected by the imaging scanning system; determining position deviations of k and j coordinates of the scan field of the manipulation scanning system and m and n coordinates of the scan field of the imaging scanning systems from each other based on the overlap; using the position deviations to compute affine transformations of the k and j coordinates of the scan field of the manipulation scanning system to the respective m and n coordinates of the scan field of the imaging scanning system for bringing the k and j coordinates of the scan field of the manipulation scanning system into line with the respective m and n coordinates of the scan field of the imaging scanning system according to an affine mapping: m=m 0 +α 11 k+α 12 j n=n 0 +α 21 k+α 22 j ; and taking into account the affine transformations in controlling at least one of the manipulation and imaging scanning systems, for at least one of: calibrating at least one of the manipulation and imaging scanning systems, and achieving pixel-precise coverage of the scan field of the manipulation scanning system with the object field of the imaging scanning system.