Improved confocal characterization of targets in nuclei of cytogenetic preparations.
OBJECTIVE
To show that cellular preparations requiring depth analysis of different domains stained by molecular cytogenetic methods (fluorescence in situ hybridization and primed in situ) can be improved by regularized factor analysis of medical image sequences (FAMIS) to isolate fluorescent probes by means of intensity depth profiles of fluorochromes, to track relevant DNA sequences (cosmids and centromeres) in cell nuclei during interphase and to improve the use of cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve probe access to their targets.


METHODS
3D sequences of images obtained by depth displacement in a confocal microscope were first analyzed by the FAMIS algorithm, which provides factor curves. Factor images then resulted from regularization methods that improve signal/noise ratio while preserving target contours.


RESULTS
Factor curves and regularized factor images helped analyze targets inside nuclei.


CONCLUSIONS
It is possible to process preparations containing numerous spots (even when they are on different planes) to differentiate stained targets, to investigate depth differences and to improve visualization and detection.
Kahn
Edmond
E
Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire Pitié-Salpêtrière, Paris, France.
Coullin
Philippe
P
Frouin
Frédérique
F
Todd-Pokropek
Andrew
A
Bernheim
Alain
A
D002467:Cell Nucleus / Q000737:chemistry*; D002478:Cells, Cultured; D002503:Centromere / Q000737:chemistry; D020732:Cytogenetic Analysis / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted