LASER-SCANNING CONFOCAL MICROSCOPY AND FACTOR-ANALYSIS OF BIOMEDICAL IMAGE SEQUENCES (FAMIS) TO DETECT AND CHARACTERIZE HPV DNA-SEQUENCES BY FISH IN HELA-CELLS

Visualisation and localisation of specific DNA sequences were performe d by fluorescence in situ hybridisation (FISH), confocal laser scannin g microscopy (CLSM), and factor analysis of biomedical image sequences (FAMIS). HeLa cells containing 10-50 copies per cell of human papillo mavirus (HPV) DNA type 18 integrated in cellular DNA were used as a mo del. HPV-DNA was identified by DNA probes and DNA-DNA hybrids mere rev ealed by alkaline phosphatase and Fast Red (FR) TR salt/naphtol-MX pho sphate. Cell nuclei were counterstained with thiazole orange (TO). FAM IS summarises image sequences into a reduced number of images called f actor images and curves called factors. Factor images correspond to sp atial distributions of the different factors. Factors estimate differe nt individual physical behaviours in the sequence (extinction velocity , spectral emission, depth emission profiles). We verified that HPV-DN A hybridisation signals are specific to the spectrum of FR, and distin guished between FR and TO. The latter result was found by taking into account differences in their extinction velocities. The focus of CLSM was improved on 3D image sequences, and the location of fluorescent si gnals inside the preparations was determined. Factor images showed tha t FR stained targets were located on different focal planes at the per iphery of the nuclei. (C) 1997 Wiley-Liss, Inc.