This study presents a method for high-precision distance measurements and for the volume-conserving segmentation of fluorescent objects with a size of the order of the microscopic observation volume. The segmentation was performed via a model-based approach, using an algorithm that was calibrated by the microscopic point spread function. Its performance was evaluated for three different fluorochromes using model images and fluorescent microspheres as test targets. The fundamental limits which the microscopic imaging process imposes on the accuracy of volume and distance measurements were evaluated in detail. A method for the calibration of the axial stepwidth of a confocal microscope is presented. The results suggest that in biological applications, 3D distances and radii of objects in cell nuclei can be determined with an accuracy of ≤ 60 nm. Using objects of different spectral signature, 3D distance measurements substantially below the lateral half width of the confocal point spread function are feasible. This is shown both theoretically and experimentally.