3-D visualization of mammalian intranuclear chromatin using computer-assisted confocal laser scan microscopy

In this study we describe three different approaches for the 3-D reconstruction of the spatial arrangement of iniranuclear chromatin. Using a Zeiss confocal laser scanning microscope (CLSM) image acquisition of optical sections was achieved in the fluorescence mode either using isolated nuclei stained with ethidiumbromide (EthBr) or on Feulgen stained tissue whole mounts. Different methods were applied for 3-D reconstruction: (i) contours of interesting structures were outlined by interactive cursor movement on a digitizer tablet (ii) digitized optical sections were transformed into image stacks by a software implemented on the microscope system and finally processed for a 3-D display and (iii) a ray-tracing method was used to provide a 3-D display of reconstructed surfaces from serial CLSM images after extensive image preprocessing. The characteristics of the different methods are discussed with respect to the biological system used.