Factor analysis of confocal image sequences of human papillomavirus DNA revealed with fast red in cervical tissue sections stained with TOTO-iodide

OBJECTIVE: To visualize and localize specific DNA sequences by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and facto r analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus (HPV) DNA was identified in cervical tis sue sections with biotinylated DNA probes recognizing the whole genome of H PV DNA types 18 and 16, and DNA-DNA hybrids were revealed by streptavidin-a lkaline phosphatase and Fast Red (FX). Cell,nuclei were counterstained with TOTO-iodide. Image sequences were obtained using successive dynamic or spe ctral sequences of images on different optical slices from CLSM. The locati on of fluorescent signals inside tissue preparations was determined by FAMI S and/or selection of filters at emission. Image sequences were summarized into a reduced number of images, called "factor images," and curves, called "factors." Factors estimate spectral patterns and depth emission? profiles . Factor images correspond to spatial distributions gf the different factor s. RESULTS: We distinguished between FR and nucleus staining in HPV DNA hybrid ization signals by taking into account differences ill their spectral patte rns and improved visualization visualization by taking into account differe nces in their focus (depth emission profiles). CONCLUSION: FAMIS, together with CLSM, made possible the detection ann char acterization of HPV DNA sequences in cells of cervical tissue sections.