Smart 3D-FISH: automation of distance analysis in nuclei of interphase cells by image processing.
BACKGROUND
Detection of fluorescent probes by fluorescence in situ hybridization in cells with preserved three-dimensional nuclear structures (3D-FISH) is useful for studying the organization of chromatin and localization of genes in interphase nuclei. Fast and reliable measurements of the relative positioning of fluorescent spots specific to subchromosomal regions and genes would improve understanding of cell structure and function.


METHODS
3D-FISH protocol, confocal microscopy, and digital image analysis were used.


RESULTS
New software (Smart 3D-FISH) has been developed to automate the process of spot segmentation and distance measurements in images from 3D-FISH experiments. It can handle any number of fluorescent spots and incorporate images of 4',6-diamino-2-phenylindole counterstained nuclei to measure the relative positioning of spot loci in the nucleus and inter-spot distance. Results from a pilot experiment using Smart 3D-FISH on ENL, MLL, and AF4 genes in two lymphoblastic cell lines were satisfactory and consistent with data published in the literature.


CONCLUSIONS
Smart 3D-FISH should greatly facilitate image processing and analysis of 3D-FISH images by providing a useful tool to overcome the laborious task of image segmentation based on user-defined parameters and decrease subjectivity in data analysis. It is available as a set of plugins for ImageJ software.
Gué
Michael
M
USM 0503, UMR 5153 CNRS-MNHN, U 565 INSERM, Département Régulations, Développement et Diversité Moléculaire, Muséum National d'Histoire Naturelle, Paris, France.
Messaoudi
Cédric
C
Sun
Jian Sheng
JS
Boudier
Thomas
T
D000465:Algorithms; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015203:Reproducibility of Results