Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect 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 (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20-50 and 1-2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction 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 stainings, 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 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.