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 factor analysis of biomedical image sequences (FAMIS).


METHODS
Human papillomavirus (HPV) DNA was identified in cervical tissue sections with biotinylated DNA probes recognizing the whole genome of HPV DNA types 18 and 16, and DNA-DNA hybrids were revealed by streptavidin-alkaline phosphatase and Fast Red (FR). Cell nuclei were counterstained with TOTO-iodide. Image sequences were obtained using successive dynamic or spectral sequences of images on different optical slices from CLSM. The location of fluorescent signals inside tissue preparations was determined by FAMIS 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 of the different factors.


RESULTS
We distinguished between FR and nucleus staining in HPV DNA hybridization signals by taking into account differences in their spectral patterns and improved visualization by taking into account differences in their focus (depth emission profiles).


CONCLUSIONS
FAMIS, together with CLSM, made possible the detection and characterization of HPV DNA sequences in cells of cervical tissue sections.
Kahn
E
E
Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire Pitié Salpêtrière, Paris. kahn@imed.jussieu.fr
Lizard
G
G
Frouin
F
F
Roignot
P
P
Chardonnet
Y
Y
Di Paola
R
R
D001391:Azo Compounds / Q000737:chemistry; D001706:Biopsy; D002467:Cell Nucleus / Q000378:metabolism / Q000473:pathology / Q000821:virology; D002584:Cervix Uteri / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis*; D005163:Factor Analysis, Statistical; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D016591:Histocytological Preparation Techniques; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D018613:Microscopy, Confocal; D027383:Papillomaviridae / Q000302:isolation & purification*; D011806:Quinolinium Compounds / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry; D002583:Uterine Cervical Neoplasms / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000378:metabolism / Q000473:pathology / Q000821:virology*
D001391:Azo Compounds; D004279:DNA, Viral; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D013844:Thiazoles; C075295:1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene)quinolinium; C005215:Fast Red S