Application of confocal laser scanning microscope on fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. However, it is difficult to obtain the characteristic or three-dimensional information of individual DNA sequence in nuclei using the conventional fluorescent microscope. Confocal Laser Scanning Microscope (CLSM) was applied on the touch preparations of ovarian neoplasia and tissue sections of melanoma, which were hybridized with centrometric or c-erb B 2 (HER-2/neu) oncogene-specific probes. It enabled us to observe clear optical sections in vertical or horizontal directions at any depth from the top to bottom of samples and also to reveal the difference of distribution of specific DNA sequences in nuclei using image-analytical devices. Copies of chromosome 6 centromeres showed an eccentric localization on optical sections, although those of c-erb B 2 distributed throughout the plans. Furthermore, the reconstructed three-dimensional images revealed that the centromere of chromosome 6 localized close to the nuclear membranes and c-erb B 2 oncogene spread sparsely, or in a dendrometric shape, throughout the nuclei. Since an application of CLSM on the samples which hybridized with DNA sequences specific probes clarified the individual localization of sequences in the nuclei of interphase, this is an especially useful method for biological dosimetry and cancer biology.