Method for imaging a sample using a fluorescence microscope with stimulated emission depletion

A method for imaging a sample using a fluorescence microscope with stimulated emission depletion, the method comprising:

• controlling the fluorescence microscope and an imaging process of the fluorescence microscope by a microscope controller such that the following steps are performed:
• generating an illumination focus by virtue of overlaying a focused depletion light distribution on a focused excitation light distribution for the stimulated emission depletion, as a result of which, during illumination with the illumination focus, the sample is excited to emit imaging fluorescence light only within an effective excitation focus, an extent of which is reduced in relation to an extent of the excitation light distribution;
• scanning a plurality of sample segments successively within a target region of the sample by the illumination focus with a first spatial resolution that has been adapted to the reduced extent of the effective excitation focus, the sample segments being imaged into a corresponding plurality of image segments, from which a scanned image is generated;
• generating an overview image of the target region with a second spatial resolution prior to the imaging process, the second spatial resolution being lower than the first spatial resolution and higher than a third spatial resolution that has been adapted to the extent of the excitation light distribution, the overview imago being analyzed such that image regions without relevant image information are identified;
• and
• reducing at least a radiant flux of the depletion light distribution within a scope of the imaging process when scanning the sample segments which are assigned to the image regions without relevant image information that were identified in the overview image.