Laser scanning microscope and its operating method

A laser scanning microscope comprising:

• manipulation and imaging scanning systems having respective scan fields defined by first and second illuminating lights independently controlled in the x- and y-directions, the manipulation scanning system being controllable to provide a stationary focus of the first illumination light on an unstructured sample for creating a reference pattern of a minimum of three different points on the unstructured sample in the scan field thereof through modification-inducing illumination of the sample by the first illumination light at a minimum of three different scan field positions,
• a beam-combiner for combining the illuminating lights of the manipulation and imaging scanning systems,
• at least one detector in the imaging scanning system for detecting an image of the reference pattern created on the sample;
• and
• means for creating an overlap of the reference pattern created by the manipulation scanning system with the image of the reference pattern detected by the at least one detector, for determining position deviations of coordinates of the scan fields of the manipulation and imaging scanning systems from each other based on the overlap, and for taking into account the coordinate 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 systems.

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 and the imaging scanning system each having a programmable, automatically controllable beam deflecting device, the method comprising the steps of:

• arranging a structured reference pattern in the form of a grid in the plane of the sample or an intermediate image plane;
• following the step of arranging of the reference pattern, detecting an image of the reference pattern with the manipulation scanning system and with the imaging scanning system independently of each other;
• creating an overlap of the image of the reference pattern detected with the manipulation scanning system with the image of the reference pattern detected with the imaging scanning system;
• determining position deviations of the image of the reference pattern detected with the manipulation scanning system from the image of the reference pattern detected with the imaging scanning system, based on the overlap;
• using the position deviations to compute coordinate transformations for bringing the images of the reference pattern detected with the manipulation scanning system and with the imaging scanning system into line with each other;
• and
• using the coordinate transformations to adjust the beam deflecting device of at least one of the manipulation scanning system and the imaging scanning system to bring the images of the reference pattern into line with each other.

A laser scanning microscope comprising:

• a structured reference pattern in the form of a grid arranged in the plane of a sample or an intermediate image plane,
• a manipulation scanning system and an imaging scanning system having respective scan fields defined by independently-controlled first and second illuminating lights for independently illuminating the reference pattern, at least one of the manipulation scanning system and the imaging scanning system having a programmable, automatically controllable beam deflecting device,
• a beam-combiner for combining the illuminating lights of the manipulation and imaging scanning systems,
• means for detecting independently from each other first and second images of the reference pattern resulting respectively from illumination of the reference pattern by the first and second illumination lights, and
• means for creating an overlap of the independently detected first and second images, for determining position deviations of coordinates of the scan fields of the manipulation and imaging scanning systems from each other based on the overlap, and for using the coordinate transformations to adjust the beam deflecting device of at least one of the manipulation scanning system and the imaging scanning system to bring the images of the reference pattern into line with each other.