Method for high-resolution 3D localization microscopy, wherein - a sample having an imaging-side boundary surface is used, - in an excitation step, the sample is illuminated with excitation radiation in order to excite fluorescence markers in the sample to emit light, - in an imaging step, the sample is imaged into a frame along an imaging direction by means of an imaging optical unit , wherein the frame contains image representations of the light-emitting fluorescence markers and the imaging optical unit has an optical resolution, - the excitation and imaging steps are repeated more than once to produce a plurality of frames , wherein the excitation steps are performed such that, at least for some of the light-emitting fluorescence markers , their image representations are isolated in each of the frames , - in each case a location statement for the corresponding fluorescence marker is ascertained from the isolated image representations of the light-emitting fluorescence markers in the plurality of frames produced, which statement has an accuracy that goes beyond the optical resolution, - a high-resolution overall image is produced from the location statements thus ascertained, characterized in that - the imaging optical unit has exactly one focal plane , - the imaging optical unit is set in a way such that the focal plane lies in the imaging direction outside the sample between the boundary surface and the imaging optical unit, and - the isolated image representations of the light-emitting fluorescence markers in the frames are analysed with respect to their size , and, from the size , a statement regarding a depth location is derived which indicates how far below the boundary surface the corresponding light-emitting fluorescence marker lies along the imaging direction. - a sample having an imaging-side boundary surface is used, - in an excitation step, the sample is illuminated with excitation radiation in order to excite fluorescence markers in the sample to emit light, - in an imaging step, the sample is imaged into a frame along an imaging direction by means of an imaging optical unit , wherein the frame contains image representations of the light-emitting fluorescence markers and the imaging optical unit has an optical resolution, - the excitation and imaging steps are repeated more than once to produce a plurality of frames , wherein the excitation steps are performed such that, at least for some of the light-emitting fluorescence markers , their image representations are isolated in each of the frames , - in each case a location statement for the corresponding fluorescence marker is ascertained from the isolated image representations of the light-emitting fluorescence markers in the plurality of frames produced, which statement has an accuracy that goes beyond the optical resolution, - a high-resolution overall image is produced from the location statements thus ascertained, characterized in that - the imaging optical unit has exactly one focal plane , - the imaging optical unit is set in a way such that the focal plane lies in the imaging direction outside the sample between the boundary surface and the imaging optical unit, and - the isolated image representations of the light-emitting fluorescence markers in the frames are analysed with respect to their size , and, from the size , a statement regarding a depth location is derived which indicates how far below the boundary surface the corresponding light-emitting fluorescence marker lies along the imaging direction. <EOS>