Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) todetect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells

Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLS M), and four-dimensional factor analysis of biomedical image sequences (4D- FAMIS). HeLa and SiHa cells containing, respectively 20-50 and 1-2 copies p er cell of human papillomavirus (HPV) DNA type is and 16 integrated in cell ular DNA were used as models, HPV-DNA was identified using DNA probes conta ining the whole genome of HPV-DNA type 18 or 16, and DNA-DNA hybrids were r evealed by alkaline phosphatase and Fast Red, Cell nuclei were counterstain ed with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtain ed using successive dynamic or spectral sequences of images on different op tical sections from CLSM, The location of fluorescent signals within the pr eparations was determined by FAMIS. This original method summarizes image s equences into a reduced number of images called factor images, and curves c alled factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emis sion profiles. Factor images correspond to spatial distributions of the dif ferent factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their e xtinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus staining s, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1-2 copies per cell of HPV-DNA type 16 locat ed in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, mad e the detection and characterization of low copy numbers of genes in whole cells possible.