CONFOCAL FLUORESCENCE ANALYSIS OF THE DEPTH AND FOCUS OF CYTOGENETIC PREPARATIONS

OBJECTIVE: To characterize differences in the depth of fluorescent pro bes, to observe estimated depth levels (focal planes) on fluorescent i n situ hybridization preparations by factor analysis of medical image sequences and to use cytogenetic techniques resulting in flat preparat ions of whole cells that are assumed to preserve the probes' access to their targets in the human nuclear interphase. STUDY DESIGN: We used labeling of the targets by the probes (sequences labeled by fluoroscei n isothiocyonate [FITC]) in the nuclei, stained by propidium iodide. T he 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 obta ining sequences of images from spherical fluorescent bends to verify { z} focusing, to visualize depth differences and to analyze estimated f actor images. RESULTS: Estimated images showed depth differences in FI TC-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. CONCLUSION: It is possible to stu dy 3-D architecture of flattened preparations and penetration of fluor ophores inside the beads and to evaluate depth differences.