A method for high-resolution 3D fluorescence microscopy, wherein fluorescence emitters in a sample are repeatedly excited to emit fluorescence, and still images are produced of the sample by means of a microscope. The microscope has an imaging beam path with an optical resolution and a focal plane, wherein the fluorescence emitters are excited to emit fluorescence in such a manner that at least a subset of the fluorescence emitters is isolated in each still image so that the images of these fluorescence emitters can be separated in the still images within the optical resolution. The positions of the fluorescence emitters are localized in the generated still images, from the images of the isolated fluorescence emitters, with a location accuracy exceeding the optical resolution, and a high-resolution composite image is generated therefrom.