Confocal fluorescence analysis of the depth and focus of cytogenetic preparations

To characterize differences in the depth of fluorescent probes, to observe estimated depth levels (focal planes) on fluorescent in situ hybridization preparations by factor analysis of medical image sequences and to use cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve the probes' access to their targets in the human nuclear interphase.

We used labeling of the targets by the probes (sequences labeled by fluoroscein isothiocyonate [FITC]) in the nuclei, stained by propidium iodide. The investigation was performed on this model by three-dimensional (3-D) sequences of images obtained on a single photomultiplier detector of a confocal microscope by selection of emission between 510 and 550 nm and by (z) displacement. The investigation was also performed by obtaining sequences of images from spherical fluorescent beads to verify (z) focusing, to visualize depth differences and to analyze estimated factor images.

Estimated images showed depth differences in FITC-stained targets as well as in nuclei, stained with propidium iodide, in interphase and in fluorescent beads, that could not be visualized by conventional 3-D reconstruction.

It is possible to study 3-D architecture of flattened preparations and penetration of fluorophores inside the beads and to evaluate depth differences.