Abstracts

Abstract

Laser confocal microscopy is a powerful tool for histochemical and cytochemical studies. It enables us to detect the fluorescencelabeled molecules in the specimens by optical. Simultaneous staining of nuclei with appropriate fluorescent dyes as counterstaining greatly facilitates the identification of the localization of the label. Recently, a variety of nucleic acid binding fluorescent dyes have been developed. We screened 20 nucleic acid-specific dyes for use in nuclear staining in laser confocal microscopy with Kr/Ar laser. Results were evaluated in 1) fluorescent intensity, 2)DNA specificity, and 3)bleaching characteristics. Among green fluorochromes excited at 488 nm, YO-PRO-1, SYTOX Green, and SYBR Green l were suitable for nuclear DNA staining. Among red fluorochromes excited at 568 nm, propidium iodide stained nuclear DNA with simultaneous staining of cytoplasmic and nucleolar RNAs. Among far-red fluorochromes excited at 647 nm,TO-PRO-3 was specific for DNA, whereas TOTO-3 strongly co-stained cytoplasmic and nucleolar RNAs. Bleaching by laser illumination was retarded by mounting with media containing and-bleaching reagents such as DABCO and PPDA. Nuclear staining fluorescent dyes should be selected by comparing these characteristics. We evaluated both the number of chromosome 17 and nuclear DNA content on an identical nucleus by means of computer-controlled auto-scanning cytofluorometry in order to demonstrate the significance of the nuclei with 17-aneusomy among whole population of tumor cells in cases of human colorectal tumor. MATERIALS and METHODS We investigated a total of 14 lesions from surgically resected colorectal tumors. We prepared isolated cell smears from paraffin-embedded archival blocks, and memorized the position of the cells using a computer-controlled auto-scanning stage. Then, we evaluated nuclear DNA content and the number of chromosome 17 sequentially in the order of cell position data. We compared statistics of DNA profile between the nuclei with 17-disomy and those with 17-aneusomy. RESULTS and DISCUSSIONSWe detected an alteration of DNA profile in 3 diploid adenomas and 3 aneuploid carcinomas. In the 3 diploid adenomas, we found a minor population of cells having DNA aneuploid in the 17-aneusomy cells, and the existence of this population resulted in the significant difference between the nuclei with 17-disomy and 17-aneusomy. In the 3 aneuploid carcinomas, there was a remarkable alteration in the height of each peak of the DNA profile. We considered that an alteration in the constituent ratio of karyotypically different subpopulations among cytofluorometrically distinct subpopulations yielded the significant difference of DNA profile.