Confocal image characterization of human papillomavirus DNA sequences revealed with Eu in HeLa cell nuclei stained with Hoechst 33342

OBJECTIVE: To visualize and localize specific viral DNA sequences revealed with Eu by fluorescence in situ hybridization, confocal laser scanning micr oscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus DNA (HPV-DNA) was identified in HeLa cel ls with biotinylated DNA probes recognizing HPV-DNA types 16/28. DNA-DNA hy brids were revealed by a three-step immunohistochemical amplification proce dure involving an antibiotin mouse monoclonal antibody, a biotinylated goat antimouse polyclonal antibody and streptavidin-Eu. Cell nuclei were counte rstained with Hoechst 33342. Image sequences were obtained using a CLSM tha t made possible ultraviolet excitation. The location of fluorescent signals inside cellular preparations was determined by FAMIS and selection of filt ers at emission. Image sequences were summarized into a reduced number of i mages, or factor images, and curves, or factors. Factors estimate spectral or temporal patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between Eu corresponding to HPV-DNA hybridization signals and nuclear staining by taking into account differences in their s pectral and temporal patterns and (using their decay rates). CONCLUSION: FAMIS, together with CLSM and Eu, made possible the detection a nd characterization of viral papillomavirus DNA sequences in HeLa cells.