Characterization of cosmids and telomeres in cytogenetic preparations by 3D confocal fluorescence.

OBJECTIVE

To analyze, with fluorescent probes, by three-dimensional (3D) emission patterns, fluorescence in situ hybridization (FISH) preparations (cosmids, telomeres) and to perform factor analysis of medical image sequences (FAMIS); to use FISH to track relevant DNA sequences in cell nuclei during interphase and in mitotic chromosomes; and to use cytogenetic techniques, resulting in flat preparations of whole cells that are assumed to preserve probe access to their targets.

STUDY DESIGN

The study design entailed labeling targets by probes (sequences labeled by fluorescein isothiocyanate) in nuclei and/or chromosomes stained by propidium iodide. Visualization of targets was improved when 3D sequences of images obtained on a single photomultiplier detector of the confocal microscope by z stepping were investigated by FAMIS.

RESULTS

Factors and factor images showed that targets could be detected and differentiated in focal planes inside nuclei or chromosomes.

CONCLUSION

It is possible to localize cosmids in cell nuclei at interphase and telomeres in mitotic chromosomes by means of 3D sequences of images.