To study the 3D-organization and 3D-pathology of the genome in intact cell nuclei, precise and accurate 3D-object localizations and 3D-distance measurements of fluorescent labelled chromatin-nanostructures are required. For this purpose, a high precision fluorescence microscope setup with spatially modulated excitation (SME) has been built up combining advantages of an epifluorescence microscope with interferometric laser illumination in the direction of the optical axis and optical sectioning. The SME allows high precision localization in the nanometer range resulting in a considerable increase of the axial resolution equivalent. This is shown for a configuration of five fluorescent microspheres of 100 nm diameter. Since the optical sections are acquired in an epifluorescent mode, image analysis procedures of high precision spectral distance microscopy were applied to determine the lateral particle localization by the position of the bary center of intensity. From these data, relative axial distances as well as relative 3D-distances were calculated. The results indicate that distance measurements between fluorescent objects can be performed with an accuracy of more than one order of magnitude better than the lateral epifluorescent optical resolution given the full width half maximum of the central peak of the effective point spread function. Since the definition of the optical resolution in refraction limited optical systems is based on distance measurement, the measure for the accuracy of our results in precision distance calculation is called resolution equivalent.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.