Three-dimensional imaging of interphase cell nuclei with laser scanning microscopy.

During the past decade 3-dimensional image processing has become an important key component in biological research mainly due to two different developments. The first is based on an optical instrument, the so-called confocal laser scanning microscope, allowing optical sectioning of the biological specimen. The second is a biological preparatory method, the so-called FISH-technique (Fluorescence-In-Situ-Hybridization), allowing labeling of certain cellular and sub-cellular compartments with highly specific fluorescent dyes. Both methods make it possible to investigate the 3-dimensional biological framework within cells and nuclei. image acquisition with confocal laser scanning microscopy must deal with different limits of resolution along and across the optical axis. Although lateral resolution is about 0.7 times better than in non-confocal arrangements, axial resolution is more than 3- 4 times poorer than that of the lateral (depending on the pinhole size). For 3D reconstruction it is desirable to improve axial resolution in order to provide nearly identical image information across the 3 dimensional specimen space. This presentation will give an overview of some of the most popular restoration and deblurring algorithms used in 3D image microscopy. After 3D image restoration, segmentation of certain details of the cell structure is usually the next step in image processing. We compared two different kinds of algorithms for segmentation of chromosome territories in interphase cell nuclei. One is based on Mathematical Morphology, the other on Split & Merge methods. The segmented image regions provided the basis for chromosome domain reconstruction as well as for regional localization for subsequent quantitative measurements. As a result the chromatin density within certain chromosome domains as well as some terminal DNA sequences (telomere signals) could be measured.