SPECTRAL AND DYNAMIC CONFOCAL FLUORESCENCE CHARACTERIZATION OF CYTOGENETIC PREPARATIONS

Investigations were performed on fluorescent in situ hybridization (FI SH) preparations to examine whether factor analysis of medical image s equences (FAMIS) can be used to isolate fluorescent probes by means of their spectral and/or extinction dynamic emission properties. FISH is used to track down chromosomes of interest in cell nuclei and mitoses . Cytogenetic techniques producing flat preparations of whole cells we re assumed to preserve the probes' access to their targets. To isolate the result of hybridization in the human nuclear interphase, we used a confocal microscope. Labelling of the targets by the probes (sequenc es labelled by FITC and TRITC) in the nuclei stained by propidium iodi de was used as a biological model. We used two methods to isolate the component parts of the model: multispectral analysis and dynamic studi es. In the case of multispectral analysis, the investigation was perfo rmed on 2D and 3D sequences of 28 images obtained on a single photomul tiplier (PM) detector of the confocal microscope by selection of emiss ion through 10-nm interference filters in the range of 500-780 nm and by z-displacement in each filter setting. In the case of dynamic studi es, the investigation was performed on sequences of 30-70 images obtai ned on the same detector by single or average integrated acquisition o f 10-30 scans. Confocal scanning yields images with constant excitatio n time. These images were investigated by FAMIS and the results reveal ed that the spectra and kinetics as factors, and factor images corresp onded to FITC and TRITC stained targets, as well as to propidium iodid e stained interphase. In conclusion, we could verify that targets were isolated through the spectrum of the fluorescent probes and could be distinguished from the propidium iodide used to stain the nuclei. It w as also possible to distinguish them from the propidium iodide by taki ng into account differences in photobleaching of the different fluoroc hromes. The study leads us to process displacements by registration me thods prior to factor analysis to improve the results.