Three-dimensional fluorescence in situ hybridization. Chromosomal studies on nuclei from cytogenetic preparations by confocal microscopy.
OBJECTIVE
To determine the three-dimensional (3D) position of target sequences and chromosomal volumes in interphase human nuclei by confocal laser scanning microscopy (CLSM) by using the heterochromatin part of the long arm of human chromosome Y (HPLAHC) as a target for specific DYZ1 probes, then by D10Z1 probes specific to the centromere of chromosome 10.


METHODS
Fluorescence in situ hybridization information inside chromosomal preparations was obtained with FITC-labelled probes and propidium iodide (PI) as a DNA-specific stain. To have a control in the experiment, HPLAHC Y was taken as a model of a domain and the centromere of chromosome 10 as a model of a single centromere spot. To have access to their 3D visualization, we selected FITC and PI patterns of fluorescence when optical slices were obtained and used a 3D reconstruction software.


RESULTS
Labelling of the target by the probes was characteristic of Y heterochromatin and chromosome 10 centromere localizations and allowed observation of their domain in the x, y and z directions.


CONCLUSIONS
This work was performed on two sets of 30 stained interphase nuclei. Deformations were confirmed by fluorescent spherical beads mounted in the same medium and scanned in the same conditions.
Kahn
E
E
Institut National de la Santé et de la Recherche Médicale U.66, Institut Gustave Roussy, Villejuif, France.
Hotmar
J
J
Bernheim
A
A
D002467:Cell Nucleus; D002478:Cells, Cultured; D002503:Centromere; D002879:Chromosomes, Human, Pair 10; D015342:DNA Probes; D006570:Heterochromatin; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007399:Interphase; D008214:Lymphocytes / Q000166:cytology; D008297:Male; D018613:Microscopy, Confocal; D014998:Y Chromosome
D015342:DNA Probes; D006570:Heterochromatin