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Characterization of cosmids and telomeres in cytogenetic preparations by 3D confocal fluorescence
1Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire Pitié-Salpêtrière, Paris.
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.