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 microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS).


METHODS
Human papillomavirus DNA (HPV-DNA) was identified in HeLa cells with biotinylated DNA probes recognizing HPV-DNA types 16/18. DNA-DNA hybrids were revealed by a three-step immunohistochemical amplification procedure involving an antibiotin mouse monoclonal antibody, a biotinylated goat antimouse polyclonal antibody and streptavidin-Eu. Cell nuclei were counterstained with Hoechst 33342. Image sequences were obtained using a CLSM that made possible ultraviolet excitation. The location of fluorescent signals inside cellular preparations was determined by FAMIS and selection of filters at emission. Image sequences were summarized into a reduced number of images, 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 spectral and temporal patterns and (using their decay rates).


CONCLUSIONS
FAMIS, together with CLSM and Eu, made possible the detection and characterization of viral papillomavirus DNA sequences in HeLa cells.
Kahn
E
E
Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire Pitié-Salpêtrière, 91 boulevard de l'Hôpital, 75634 Paris, France. kahn@imed.jussieu.fr
Lizard
G
G
Frouin
F
F
Souchier
C
C
Bernengo
J C
JC
Siitari
H
H
Cuenod
C A
CA
Clément
O
O
Frija
G
G
Gambert
P
P
Todd-Pokropek
A
A
D001483:Base Sequence; D001562:Benzimidazoles; D019921:Biotinylation; D002467:Cell Nucleus / Q000378:metabolism / Q000821:virology*; D015344:DNA Probes, HPV / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D005063:Europium; D006367:HeLa Cells; D016591:Histocytological Preparation Techniques; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D027383:Papillomaviridae / Q000235:genetics*; D011851:Radioactivity; D011868:Radioisotopes; D013057:Spectrum Analysis; D013194:Staining and Labeling; D013997:Time Factors
D001562:Benzimidazoles; D015344:DNA Probes, HPV; D004279:DNA, Viral; D011868:Radioisotopes; D005063:Europium; C017807:bisbenzimide ethoxide trihydrochloride