High-resolution
microscopy method for the three-dimensional position determination of objects, in particular individual fluorophores, preferably for spatially high-resolution
luminescence microscopy of a sample (7) which is labelled with labeling molecules (1) which can be activated or switched with a
signal such that they can only be excited to emit certain
luminescence radiation in the activated state, wherein the method comprises the following steps: a) Introducing the
signal onto the sample (7) such that only a subset of the labeling molecules (1) present in the sample (7) are activated, wherein the sample (7) contains subregions in which activated labeling molecules (1) are at least as far apart from the nearest neighboring activated labeling molecules (1) as is greater than or equal to a length which results from a predetermined
optical resolution, b) Excitation of the activated molecules (1) to emit
luminescence radiation, c) Detection of the luminescence
radiation with the predetermined
optical resolution, and d) Generating a
single image from the luminescence radiation recorded in step c), wherein the geometric locations of the luminescence-emitting labeling molecules are determined with a spatial resolution increased above the predetermined
optical resolution, where the steps are repeated several times and the resulting individual images are combined to form a complete image, characterized by the fact that an object is imaged via an imaging
system (OB, TL) with a
microscope objective (15, OB) onto an
area detector (5, CCD) consisting of individual
detector elements, wherein at least one
microlens arrangement (ML, ZL), which is partially located in front of the
detector elements, images a different object plane (OE1) onto the
detector elements in the direction of light behind the microlenses than onto detector elements in front of which there are no microlenses.