Abstract: Chromosomes play a fundamental role in heredity. This reasons the interest in new highly resolving microscopical techniques for their analysis. New preparation techniques have offered a direct approach to detect specific nucleic acid sequences by in situ hybridization. Labelled DNA probes detected by fluorochrome conjugates make it possible to visualize regions down to single genes by light microscopy. Scanning force microscopy (SFM) provides the possibility to image surfaces of biological objects with a resolution one to two orders of magnitude better than a classical fluorescence light microscope. Here, air dried human metaphase chromosomes were examined by SFM before and after in situ hybridization. Different hybridization protocols were compared in their influence on chromosomal morphology. An immunogold technique was introduced for topographic labeling detection by SFM. By propidium iodide staining, identically the same chromosomes which were already examined by SFM were visualized by high resolution confocal light microscopy. The SFM results suggest that the hybridization procedure induced alterations in the overall chromosomal morphology which were not directly detectable by the fluorescence image in light microscopy. Using the immunogold labelling technique and silver enhancement, it was possible to study hybridization features and chromosomal morphology at high resolution simultaneously by SFM. The application of this approach may offer possibilities to investigate the hybridization mechanisms and to develop new hybridization protocols inducing minimal ultrastructural effects on the chromosomes. !32
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